CD38 antibody

ABSTRACT

The present disclosure provides antibody sequences found in antibodies that bind to human CD38. In particular, the present disclosure provides sequences of anti-human CD38 antibodies. Antibodies and antigen-binding portions thereof including such sequences present features compatible with pharmaceutical manufacturing and development can be provided as fully human antibodies (e.g., fully human monoclonal antibodies or antigen-binding fragments) that can be useful for medical methods and compositions, in particular for treating cancer.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is the U.S. national phase application filed under 35U.S.C. § 371 claiming benefit to International Patent Application No.PCT/EP2018/072272, filed on Aug. 16, 2018, which claims priority to U.S.Provisional Patent Application No. 62/546,336, filed Aug. 16, 2017, andU.S. Provisional Patent Application No. 62/582,609, filed Nov. 7, 2017,the disclosures of which are incorporated herein by reference in theirentirety.

BACKGROUND

CD38 is a type a type II membrane receptor glycoprotein having enzymaticactivities, in particular as an important ADP-ribosyl cyclase thatproduces cyclic adenosine diphosphate ribose (cADPR) from nicotinamideadenine dinucleotide. CD38 can also hydrolyze NAD+ into ADPR. Generationof ADPR may modulate immunological responses as it is upstream of apathway leading to the production of immune-suppressive adenosine.Different extracellular stimuli can induce cADPR production. cADPR isimportant to the mobilization of intracellular calcium stock that isinvolved in many cell functions such as cell proliferation,differentiation, adhesion, and signal transduction. CD38 was initiallyidentified as a leukocyte activation marker but plays dual roles asreceptor and ectoenzyme, endowed with cell signalling and cellhomeostasis activities. CD38 has been linked to various human diseases,including malignancies such as chronic lymphocytic leukemia, myeloma andovarian carcinoma (Quarona V, et al., 2013; Wei W, et al., 2014).

CD38 is found on the surface of many cell types that are involved inimmunological responses (in short referred to as immune cells),including effector cells such as T and B lymphocytes and NK cells, butalso immune suppressive cells such as regulatory T and B cells, myeloidderived suppressive cells (MDSCs) or tumour associated macrophages(Chevrier S et al. 2017). For instance, in lung cancer patients,anti-PD-1 treatment induced proliferation of PD-1 expressing T cellsthat expressed high levels of CD38 (Kamphorst A O et al., 2017). Theimportance of cADPR- and CD38-mediated Ca2+ signalling for biologicalactivity of immune cells, in particular for the modulation of the immuneresponse in physiological and pathological conditions, has beendescribed in the literature (Morandi F et al., 2015; Rah S Y et al.,2015).

CD38 is highly expressed by cancer cells in multiple myeloma patients atall stages of disease and in CLL patients with a poor prognosis. VariousCD38-targeting therapies are developed by generating compounds that actmainly as CD38 antagonists or inhibitors (de Weers M et al., 2011; vande Donk N W et al., 2016; Horenstein A L et al., 2017). Anti-CD38monoclonal antibodies acting as CD38 agonists (such as the one namedIB4) have also been characterized as inducing the mobilization ofcalcium ions, CD38 shedding, NK cell-mediated cytotoxicity, cytokinesecretion (in particular Interleukin 6 and Interferon gamma), andproliferation of human T lymphocytes, among other activities, and weremodified to deliver immunotoxins (Malavasi F et al., 2008; Hara-YokoyamaM et al., 2008; Frasca L et al, 2006; Karakasheva T et al., 2015). Sucha positive effect on immune cells may be related to induction of Ca2+mobilization, inhibition of CD38 enzymatic activity, and/or activationof intra-cellular signaling pathways.

Monoclonal antibodies were developed for targeted, direct killing ofCD38-expressing tumor cells and have shown promising results in theclinic. However, the activity of such anti-CD38 antibodies may berestricted to tumors in which CD38 is highly expressed on the surface ofcancer cells. In solid tumors, the expression of CD38 is generally loweror absent on the tumor cells and may be associated with tumorinfiltrating immune cells, both effector and suppressive. Therefore,there is still a need for anti-CD38 antibodies presenting activitiesthat result from the combination of different components, such asCD38-specific agonistic or modulating properties together with targetedcell killing or activation, and compatibility with pharmaceuticaldevelopment, and that can be exploited for treating cancer, inparticular for treating solid and haematological cancers.

SUMMARY

In some embodiments, the present invention provides new CD38 ModulatingAntibody Agents. In some embodiments, provided CD38 Modulating AntibodyAgents are antibodies or antigen-binding fragments that specificallybind to CD38, and particularly to human CD38, in many embodiments to asite in the human CD38 extracellular domain.

In some embodiments, provided antibodies or antigen binding fragmentsmodulate one or more features of CD38. That is, in some embodiments,level and/or activity of CD38, and/or one or more downstream effectsthereof, is detectably altered when a provided antibody is present ascompared with when it is absent. Alternatively or additionally, in someembodiments, level and/or activity of CD38, and/or one or moredownstream effects thereof, when a provided antibody is present, iscomparable to or greater than that observed under comparable conditionswhen a reference CD38 Modulating Antibody Agent (e.g., a referenceanti-CD38 antibody, such as IB-4, with a known desirable attribute;e.g., a known ability to agonize one or more features of CD38).

In many embodiments, one or more features of CD38 is enhanced when aprovided CD38 Modulating Antibody Agent (e.g., anti-CD38 antibody orantigen-binding fragment thereof) is present. For example, in someembodiments, presence of a provided CD38 Modulating Antibody Agent(e.g., anti-CD38 antibody or antigen-binding fragment thereof)correlates with increased immune cell activation, and/or proliferation.Thus, provided CD38 Modulating Antibody Agents are often referred toherein as “agonists”. Those skilled in the art, however, will appreciatethat teachings of the present disclosure are not limited by particularmechanism of action of provided antibodies or antigen-binding fragmentsthereof. Relevant structural and/or functional features of providedantibodies are described herein and speak for themselves.

In some embodiments, provided CD38 Modulating Antibody Agents (e.g.,CD38 antibodies or antigen-binding fragments) may be characterized, forexample, by effects on certain immune effector cells (e.g., NK cellsand/or T cells). Alternatively or additionally, in some embodiments,provided CD38 Modulating Antibody Agents (e.g., CD38 antibodies orantigen-binding fragments) may be characterized, for example, by effectson immune suppressive cells. For example, in some embodiments, providedCD38 Modulating Antibody Agents display activating properties withrespect to immune effector cells such as NK cells and T cells and/orcytotoxic properties towards CD38 high expressing cells such as immunesuppressive cells or tumor cells (e.g., in each case, that express CD38on their surfaces). Alternatively or additionally, in some embodiments,provided CD38 Modulating Antibody Agents are characterized by one ormore features that are associated with binding to a specific epitope inhuman CD38 extracellular domain and/or that render them particularlyamenable to pharmaceutical use and/or manufacturing.

Provided technologies, including provided CD38 Modulating AntibodyAgents (e.g., provided antibodies or antigen-binding fragments thereof(or variants of the same), compositions including them, and/or uses forthem, are useful in medicine. In some embodiments, such providedtechnologies are useful in cancer therapy and/or prophylaxis.

In some embodiments, provided CD38 Modulating Antibody Agents areexemplified by the antibodies having the sequence of aCD38-a-309, andmore in general antibodies or agents that are or comprise one or moreantigen-binding fragments or portions thereof, for example that comprisethe aCD38-a-309-HCDR3 amino acid sequence (SEQ ID NO: 3) as variableheavy chain complementarity determining region 3, and/or, in someembodiments, comprise one or both of the aCD38-a-309 HCDR1 (SEQ IDNO: 1) and HCDR2 (SEQ ID NO: 2) sequences, and/or that compete withaCD38-a-309 for binding human CD38 extracellular domain. In someembodiments, provided antibodies or antigen-binding fragments thereofbind to human CD38 with a Kd of in the 10⁻⁸M range, or below (in the10⁻⁹M range), preferably the antibodies or antigen-binding fragmentsthereof bind to human CD38 with a Kd in the 10⁻⁸ M to 10⁻¹¹ M range. Insome embodiments the Kd is from 10 to 10⁻¹¹. The Kd to evaluate thebinding affinity of the antibodies or antigen binding fragments thereofcan be obtained by standard methodologies including surface plasmonresonance (SPR) such as Biacore analysis or analysis using Forte BioOctet Systems.

In some embodiments, provided CD38 Modulating Antibody Agents (e.g.,provided antibodies or antigen-binding fragments thereof) bind to anepitope on human CD38 that is bound by aCD38-a-309. In some embodiments,such provided CD38 Modulating Antibody Agents may bind to human CD38extracellular domain. In some embodiments, provided CD38 ModulatingAntibody Agents may bind to an epitope of CD38 (e.g., when assessedusing one or more assays as described herein or otherwise known in theart). In some embodiments, provided antibodies or antigen-bindingfragments thereof may bind to human and Cynomolgus Monkey CD38 (e.g., toan extracellular epitope on human and Cynomolgus Monkey CD38) with Kdvalue in the 10⁻⁸M range, antigen-binding fragments thereof bind tohuman CD38 with a Kd of in the range of 10⁻⁸ to 10⁻¹¹ M.

In some embodiments, the provided antibodies or antigen-bindingfragments thereof do bind to a mutant human CD38 (as compared tonon-mutant human CD38 (SEQ ID NO: 9)), wherein in the mutant human CD38,the serine residue in position 274 has been substituted with aphenylalanine.

In some embodiments, the provided antibodies or antigen-bindingfragments thereof do not bind to a mutant human CD38 or binds with areduced affinity to a mutant human CD38 (as compared to non-mutant humanCD38 (SEQ ID NO: 9)), wherein in the mutant human CD38, the aspartateresidue in position 202 has been substituted with a glycine residue.

In some embodiments, the provided antibodies or antigen-bindingfragments thereof do bind to a mutant human CD38 (as compared tonon-mutant human CD38 (SEQ ID NO: 9)), wherein in the mutant human CD38,the serine residue in position 274 has been substituted with aphenylalanine and do not bind to a mutant human CD38, or binds with areduced affinity to a mutant human CD38, wherein in the mutant humanCD38 the aspartate residue in position 202 has been substituted with aglycine residue.

Among other things, the present disclosure provides a procedure (FIG. 1)that can be utilized to identify and/or characterize particularly usefulCD38 Modulating Antibody Agents (e.g., anti CD38 antibodies orantigen-binding fragments thereof) as described herein (e.g., anti-CD38antibodies or antigen-binding fragments thereof characterized by certainstructural and/or functional features, such as specific binding to humanCD38 (e.g., to an extracellular epitope thereof), inclusion of one ormore CDR sequence elements as described herein (and particularlyinclusion of an HCDR3 sequence element, optionally in combination withHCDR1 and/or HCDR2 elements), cell activating activity as describedherein, cytotoxic activity as described herein (e.g., with respect toimmune regulatory cells with relatively high levels of CD38 on theirsurfaces), and combinations thereof). In some embodiments, particularlyuseful anti-CD38 antibodies as described herein are characterized by aplurality of such features. In some embodiments, one or more antibodiesdescribed herein may be characterized as a CD38 Modulating AntibodyAgent.

Thus, as exemplified herein, certain antibodies and/or antigen-bindingfragments comprising aCD38-a-309 sequences (in particularaCD38-a-309-HCDR3 (SEQ ID NO: 3) and/or aCD38-a-309-LCDR3 (SEQ ID NO:7)) are characterized by such desirable structural and/or functionalfeatures; such antibodies and/or antigen-binding fragments thereof maybe referred to herein as CD38 Modulating Antibody Agents. Additionally,in accordance with the present disclosure, antibodies andantigen-binding fragments thereof compete with aCD38-a-309 may beparticularly useful antibodies; such antibodies and/or antigen-bindingfragments thereof may also be referred to herein as CD38 ModulatingAntibody Agents.

Antibodies (and/or antigen-binding fragments thereof) described hereinmay be particularly useful in medicine (e.g., in therapy and/or inprophylaxis, for example in the treatment of cancer), and/or for usewith respect to methods that require or involve targeting an epitopewithin human CD38 extracellular domain. Provided antibodies orantigen-binding fragments thereof may be prepared as presenting the mostappropriate isotype, in particular human isotype from the groupconsisting of IgG1, IgG2, IgG3, and IgG4 isotype antibodies, moreparticularly human IgG1.

In one aspect, the present invention provides aCD38-a-309-HCDR3 aminoacid sequence (SEQ ID NO: 3) and polypeptides that include it, such as,for example, antibodies or antigen-binding fragments comprising theaCD38-a-309-HCDR3 amino acid sequence (SEQ ID NO: 3) as variable heavychain complementarity determining region 3. In some embodiments, suchantibody or antigen-binding fragment may be further characterized bycomprising further aCD38-a-309 amino acid sequence elements such as:

a) aCD38-a-309-HCDR1 amino acid sequence (SEQ ID NO: 1) as variableheavy chain complementarity determining region 1; and/or

b) aCD38-a-309-HCDR2 amino acid sequence (SEQ ID NO: 2) as variableheavy chain complementarity determining region 2.

In some embodiments, provided antibodies or antigen-binding fragmentsthereof may comprise variable heavy chain complementarity determiningregions defined above (i.e. aCD38-a-309 amino acid sequence elements)further in the correct order, specifically separated by antibody framesequences, such the one included in aCD38-a-309-HCDR123 amino acidsequence (SEQ ID NO: 4), in particular for exerting correctly theirbinding and functional properties. For example, in some embodiments, aprovided antibody or antigen-binding fragment said thereof can compriseaCD38-a-309-HCDR123 amino acid sequence (SEQ ID NO: 4 or the HCDR1,HCDR2 and HCDR3 sequences thereof) and, optionally:

a) aCD38-a-309-LCDR1 amino acid sequence (SEQ ID NO: 5) as variablelight chain complementarity determining region 1;

b) aCD38-a-309-LCDR2 amino acid sequence (SEQ ID NO: 6) as variablelight chain complementarity determining region 2; and

c) aCD38-a-309-LCDR3 amino acid sequence (SEQ ID NO: 7) as variablelight chain complementarity determining region 3.

Thus, in some embodiments, the present invention provides an isolatedantibody or antigen-binding fragments thereof comprising a variableheavy chain comprising aCD38-a-309-HCDR123 amino acid sequence (SEQ IDNO: 4). Preferably, such isolated antibody or antigen-binding fragmentsthereof further comprises a variable light chain comprisingaCD38-a-309-LCDR123 amino acid sequence (SEQ ID NO: 8), as described inthe Examples.

In some embodiments the present invention provides an isolated antibodyor antigen-binding fragments thereof comprising a variable heavy chaincomprising the sequence:

(SEQ ID NO: 12) FTFSSYSMNWVRQAPGKGLEWVSSISSSSNYIYYADSVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCAREPIYTSGWPFDIand/or a variable light chain sequence comprising the sequence:

(SEQ ID NO: 13) RASQSVSSYLAWYQQKPGQAPRLLIYDASNRATGIPARFSGSGSGTDFTLTISSLEPEDFAVYYCQQHVNFPWT.

In some embodiments the variable heavy chain sequence of aCD38-a-309comprises the sequence:

(SEQ ID NO: 10) EVQLVESGGGLVKPGGSLRLSCAASGFTFSSYSMNWVRQAPGKGLEWVSSISSSSNYIYYADSVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCAREPIYTSGWPFDIWGQGTMVIVSSand the variable light chain sequence of aCD38-a-309 comprises thesequence:

(SEQ ID NO: 11) EIVLTQSPATLSLSPGERATLSCRASQSVSSYLAWYQQKPGQAPRLLIYDASNRATGIPARFSGSGSGTDFTLTISSLEPEDFAVYYCQQHVNFPW TFGGGTKVEIK.

The present invention also provides an antibody or antigen-bindingfragment thereof comprising the sequence of aCD38-a-309-HCDR3 (SEQ IDNO: 3) as an HCDR3 and the sequence of aCD38-a-309-LCDR3 (SEQ ID NO: 7)as an LCDR3.

The present invention also provides variant antibodies and antigenbinding fragments thereof that have certain % identities relative to areference sequence, such as a CDR sequence or a heavy or light chainvariable sequence of aCD38-a-309. Such antibodies and antigen bindingfragments thereof may also be referred to herein as CD38 ModulatingAntibody Agents.

For example, in some embodiments the anti-CD38 antibody or antigenbinding fragment thereof comprises a variable heavy chain sequencecomprising an amino acid sequence having at least 90% sequence identityto SEQ ID NO: 4. In some embodiments the anti-CD38 antibody or antigenbinding fragment thereof comprises a variable heavy chain sequencecomprising an amino acid sequence having at least 95% sequence identityto SEQ ID NO: 4. In some embodiments the anti-CD38 antibody or antigenbinding fragment thereof comprises a variable heavy chain sequencecomprising an amino acid sequence having at least 98% sequence identityto SEQ ID NO: 4. In some embodiments the anti-CD38 antibody or antigenbinding fragment thereof comprises a variable heavy chain sequencecomprising the amino acid sequence of SEQ ID NO: 4.

In some embodiments the anti-CD38 antibody or antigen binding fragmentthereof comprises a variable light chain sequence comprising an aminoacid sequence having at least 90% sequence identity to SEQ ID NO: 8. Insome embodiments the anti-CD38 antibody or antigen binding fragmentthereof comprises a variable light chain sequence comprising an aminoacid sequence having at least 95% sequence identity to SEQ ID NO: 8. Insome embodiments the anti-CD38 antibody or antigen binding fragmentthereof comprises a variable light chain sequence comprising an aminoacid sequence having at least 98% sequence identity to SEQ ID NO: 8. Insome embodiments the anti-CD38 antibody or antigen binding fragmentthereof comprises a variable light chain sequence comprising the aminoacid sequence of SEQ ID NO: 8.

In some embodiments the anti-CD38 antibody or antigen binding fragmentthereof comprises a variable heavy chain sequence comprising an aminoacid sequence having at least 90% sequence identity to SEQ ID NO: 4 anda variable light chain sequence comprising an amino acid sequence havingat least 90% sequence identity to SEQ ID NO: 8. In some embodiments theanti-CD38 antibody or antigen binding fragment thereof comprises avariable heavy chain sequence comprising an amino acid sequence havingat least 95% sequence identity to SEQ ID NO: 4 and a variable lightchain sequence comprising an amino acid sequence having at least 95%sequence identity to SEQ ID NO: 8. In some embodiments the anti-CD38antibody or antigen binding fragment thereof comprises a variable heavychain sequence comprising an amino acid sequence having at least 98%sequence identity to SEQ ID NO: 4 and a variable light chain sequencecomprising an amino acid sequence having at least 98% sequence identityto SEQ ID NO: 8. In some embodiments the anti-CD38 antibody or antigenbinding fragment thereof comprises a variable heavy chain sequencecomprising the amino acid sequence of SEQ ID NO: 4 and a variable lightchain sequence comprising the amino acid sequence of SEQ ID NO: 8.

In some embodiments the anti-CD38 antibody or antigen binding fragmentthereof comprises a variable heavy chain sequence comprising an aminoacid sequence having at least 90% sequence identity to SEQ ID NO: 10. Insome embodiments the anti-CD38 antibody or antigen binding fragmentthereof comprises a variable heavy chain sequence comprising an aminoacid sequence having at least 95% sequence identity to SEQ ID NO: 10. Insome embodiments the anti-CD38 antibody or antigen binding fragmentthereof comprises a variable heavy chain sequence comprising an aminoacid sequence having at least 98% sequence identity to SEQ ID NO: 10. Insome embodiments the anti-CD38 antibody or antigen binding fragmentthereof comprises a variable heavy chain sequence comprising the aminoacid sequence of SEQ ID NO: 10.

In some embodiments the anti-CD38 antibody or antigen binding fragmentthereof comprises a variable light chain sequence comprising an aminoacid sequence having at least 90% sequence identity to SEQ ID NO: 11. Insome embodiments the anti-CD38 antibody or antigen binding fragmentthereof comprises a variable light chain sequence comprising an aminoacid sequence having at least 95% sequence identity to SEQ ID NO: 11. Insome embodiments the anti-CD38 antibody or antigen binding fragmentthereof comprises a variable light chain sequence comprising an aminoacid sequence having at least 98% sequence identity to SEQ ID NO: 11. Insome embodiments the anti-CD38 antibody or antigen binding fragmentthereof comprises a variable light chain sequence comprising the aminoacid sequence of SEQ ID NO: 11.

In some embodiments the anti-CD38 antibody or antigen binding fragmentthereof comprises a variable heavy chain sequence comprising an aminoacid sequence having at least 90% sequence identity to SEQ ID NO: 10 anda variable light chain sequence comprising an amino acid sequence havingat least 90% sequence identity to SEQ ID NO: 11. In some embodiments theanti-CD38 antibody or antigen binding fragment thereof comprises avariable heavy chain sequence comprising an amino acid sequence havingat least 95% sequence identity to SEQ ID NO: 10 and a variable lightchain sequence comprising an amino acid sequence having at least 95%sequence identity to SEQ ID NO: 11. In some embodiments the anti-CD38antibody or antigen binding fragment thereof comprises a variable heavychain sequence comprising an amino acid sequence having at least 98%sequence identity to SEQ ID NO: 10 and a variable light chain sequencecomprising an amino acid sequence having at least 98% sequence identityto SEQ ID NO: 11. In some embodiments the anti-CD38 antibody or antigenbinding fragment thereof comprises a variable heavy chain sequencecomprising the amino acid sequence of SEQ ID NO: 10 and a variable lightchain sequence comprising the amino acid sequence of SEQ ID NO: 11.

In some embodiments the anti-CD38 antibody or antigen binding fragmentthereof comprises a variable heavy chain sequence comprising an aminoacid sequence having at least 90% sequence identity to SEQ ID NO: 12. Insome embodiments the anti-CD38 antibody or antigen binding fragmentthereof comprises a variable heavy chain sequence comprising an aminoacid sequence having at least 95% sequence identity to SEQ ID NO: 12. Insome embodiments the anti-CD38 antibody or antigen binding fragmentthereof comprises a variable heavy chain sequence comprising an aminoacid sequence having at least 98% sequence identity to SEQ ID NO: 12. Insome embodiments the anti-CD38 antibody or antigen binding fragmentthereof comprises a variable heavy chain sequence comprising the aminoacid sequence of SEQ ID NO: 12.

In some embodiments the anti-CD38 antibody or antigen binding fragmentthereof comprises a variable light chain sequence comprising an aminoacid sequence having at least 90% sequence identity to SEQ ID NO:.13 Insome embodiments the anti-CD38 antibody or antigen binding fragmentthereof comprises a variable light chain sequence comprising an aminoacid sequence having at least 95% sequence identity to SEQ ID NO: 13. Insome embodiments the anti-CD38 antibody or antigen binding fragmentthereof comprises a variable light chain sequence comprising an aminoacid sequence having at least 98% sequence identity to SEQ ID NO: 13. Insome embodiments the anti-CD38 antibody or antigen binding fragmentthereof comprises a variable light chain sequence comprising the aminoacid sequence of SEQ ID NO: 13.

In some embodiments the anti-CD38 antibody or antigen binding fragmentthereof comprises a variable heavy chain sequence comprising an aminoacid sequence having at least 90% sequence identity to SEQ ID NO: 12 anda variable light chain sequence comprising an amino acid sequence havingat least 90% sequence identity to SEQ ID NO: 13. In some embodiments theanti-CD38 antibody or antigen binding fragment thereof comprises avariable heavy chain sequence comprising an amino acid sequence havingat least 95% sequence identity to SEQ ID NO: 12 and a variable lightchain sequence comprising an amino acid sequence having at least 95%sequence identity to SEQ ID NO: 13. In some embodiments the anti-CD38antibody or antigen binding fragment thereof comprises a variable heavychain sequence comprising an amino acid sequence having at least 98%sequence identity to SEQ ID NO: 12 and a variable light chain sequencecomprising an amino acid sequence having at least 98% sequence identityto SEQ ID NO: 13. In some embodiments the anti-CD38 antibody or antigenbinding fragment thereof comprises a variable heavy chain sequencecomprising the amino acid sequence of SEQ ID NO: 12 and a variable lightchain sequence comprising the amino acid sequence of SEQ ID NO: 13.

Such variant antibodies and antigen binding fragments thereof may retainor exhibit the same (or substantially the same) functional andpharmacological properties as described for the antibodies and antigenbinding fragments thereof having the heavy and light chain variablesequences disclosed herein for aCD38-a-309.

Moreover, aCD38-a-309 amino acid sequences also refer to antibodysequences that are defined by the number of substitution with respect tothe aCD38-a-309 amino acid sequence elements defined above. For example,such sequence may comprise, as variable heavy chain complementaritydetermining region 3 (HCDR3) a sequence containing up to 1, 2, 3, or 4amino acid substitutions within aCD38-a-309-HCDR3 (SEQ ID NO: 3). In afurther embodiment, aCD38-a-309 amino acid sequences also refer toantibody sequences comprising, as variable heavy chain complementaritydetermining regions 1, 2 and 3 (HCDR1, HCDR2, and HCDR3) a sequencecontaining up to 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 amino acidsubstitutions within aCD38-a-309-HCDR1, aCD38-a-309-HCDR2, andaCD38-a-309-HCDR3, and more preferably a sequence containing up to 1, 2,3, 4, 5, 6, 7, 8, 9 or 10 amino acid substitutions withinaCD38-a-309-HCDR123 (SEQ ID NO: 4) or within SEQ ID NO: 10 or SEQ IDNO:12. In some embodiments aCD38-a-309 amino acid sequences also referto antibody sequences comprising as a variable heavy chain sequence asequence containing up to 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10, amino acidsubstitutions within the framework regions of the variable heavy chainsequence. The antibodies presenting such aCD38-a-309 amino acid sequenceelements and such substitutions can still present the binding and/orfunctional properties of aCD38-a-309, and of a CD38 Modulating AntibodyAgent in general.

Such aCD38-a-309 amino acid sequences may also comprise, as variablelight chain complementarity determining region 3 (LCDR3) a sequencecontaining up to 1, 2, 3, or 4, amino acid substitutions withinaCD38-a-309-LCDR3 (SEQ ID NO: 5). In a further embodiment, aCD38-a-309amino acid sequences also refer to antibody sequences comprising, asvariable light chain complementarity determining regions 1, 2 and 3(LCDR1, LCDR2, and LCDR3) a sequence containing up to 1, 2, 3, 4, 5, 6,7, 8, 9, or 10, amino acid substitutions within aCD38-a-309-LCDR1,aCD38-a-309-LCDR2, and aCD38-a-309-LCDR3, and more preferably a sequencecontaining up to 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10, amino acidsubstitutions within aCD38-a-309-LCDR123 (SEQ ID NO: 8), or within SEQID NO: 11 or SEQ ID NO: 13. In some embodiments aCD38-a-309 amino acidsequences also refer to antibody sequences comprising as a variablelight chain sequence a sequence containing up to 1, 2, 3, 4, 5, 6, 7, 8,9, or 10, amino acid substitutions within the framework regions of thevariable light chain sequence. The antibodies presenting suchaCD38-a-309 amino acid sequence elements and such substitutions canstill present the binding and/or functional properties of aCD38-a-309,and of a CD38 Modulating Antibody Agent in general.

Accordingly, in one embodiment, the present invention provides ananti-CD38 Antibody Agent (i.e. an antibody or antigen-binding fragmentthereof and variants thereof as described herein) comprising:

-   -   a. the variable heavy chain region sequence of aCD38-a-309 (or a        variant thereof, such as an affinity matured variant thereof) or        a variable heavy chain region sequence having up to 5 amino acid        substitutions compared to the variable heavy chain region        sequence of aCD38-a-309 (or a variant thereof, such as an        affinity matured variant thereof); and/or    -   b. the variable light chain region sequence of aCD38-a-309 (or a        variant thereof, such as an affinity matured variant thereof) or        a variable light chain region sequence having up to 5 amino acid        substitutions compared to the variable light chain region        sequence of aCD38-a-309 (or a variant thereof, such as an        affinity matured variant thereof).

The aCD38-a-309 heavy chains that may incorporate amino acidsubstitutions include SEQ ID NOs 4, 10, 12. The aCD38-a-309 light chainsthat may incorporate amino acid substitutions include SEQ ID Nos 8, 11and 13.

The amino acid substitutions preferably do not adversely effect, or notsubstantially adversely effect, the functional properties of theantibodies. The substitutions may therefore be considered conservativeamino acid substitutions. Preferably, when amino acid substitutions dooccur, they occur in a ratio of 1:1, such that the total length of theheavy and/or light chain variable region does not change.

The invention also provides antibodies or antigen-binding fragmentsthereof, wherein any methionine in the light or heavy chains of theantibodies may be altered, for example to reduce methionine oxidation.For example, a methionine residue may be altered to replace it with adifferent amino acid, for example leucine or phenylalanine. Suchantibodies that have been modified in this may way need to undergofurther modification (for example affinity maturation) before arrivingat a final sequence.

In one embodiment of the invention, there is provided a variant antibodyhaving CDR1, CDR2 and CDR3 sequences of an antibody as disclosed herein(for example the CDR1, CDR2 and CDR3 sequences of aCD38-a-309), or thevariable heavy and variable light chain of any antibody as disclosedherein (for example the variable heavy and variable light chain ofaCD38-a-309), but differing from the specified sequence in that at leastone amino acid (for example a methionine) in the CDR has been changed toa different amino acid. The disclosed variants may be used andformulated as described for aCD38-a-309.

The invention also provides affinity matured antibodies, for exampleaffinity matured variants derived from any of the antibodies disclosedherein. The disclosed affinity matured variants may be used andformulated as described for aCD38-a-309.

In some embodiments the invention provides a method of preparing ananti-CD38 antibody comprising providing an antibody as herein described(e.g., aCD38-a-309 or an antigen binding fragment or variant thereof),and subjecting the antibody to affinity maturation, wherein the antibodyproduced binds to CD38 with greater affinity than the parental antibody.Preferably the produced antibody binds to CD38 with at least 20%, atleast 30%, at least 40%, more preferably at least 50% greater affinitythan the parental antibody binds to CD38, for example as measured by theKd. Methods for measuring affinity are known in the art and described inthe Examples below. The affinity matured antibodies produced by suchmethods can be formulated and used as described herein for the otheranti-CD38 Antibody Agents.

Affinity maturation may be carried out according to any suitable methodknown to the skilled person. For example, in vitro antibody displaysystems are widely used for the generation of specific antibodies withhigh affinity. In these systems, the phenotype (i.e., the antibodyfragment) is coupled to the genotype (i.e., the antibody gene) allowingthe direct determination of the sequence of the antibody. Severalsystems have been developed to achieve display of antibody repertoiresto allow subsequent selection of binders and by increasing thestringency of selection allows for the selection of higher and higheraffinity variants. The antibody fragments can be expressed in yeast,ribosomes, phage display particles or by direct coupling to DNA.

Current antibody affinity maturation methods belong to two mutagenesiscategories: stochastic and non-stochastic. Error-prone polymerase chainreaction (PCR), mutator bacterial strains, and saturation mutagenesisare typical examples of stochastic mutagenesis methods. Non-stochastictechniques often use alanine-scanning or site-directed mutagenesis togenerate limited collections of specific variants. In addition,shuffling approaches to obtain shuffled variants of the parent antibodycan also be used to improve antibodies affinity further.

Accordingly, in one embodiment of the invention, the method of affinitymaturation is selected from the group consisting of stochasticmutagenesis (for example error-prone polymerase chain reaction (PCR),mutator bacterial strains, or saturation mutagenesis), non-stochasticmutagenesis (for example alanine-scanning or site-directed mutagenesis),shuffling (for example DNA shuffling, chain shuffling or CDR shuffling)and the use of the CRISPR-Cas9 system to introduce modifications.

Affinity maturation methods are described in, for example, Rajpal etal., Proc Natl Acad Sci USA, 2005, 102(24):8466-71, Steinwand et al.,MAbs, 2014, 6(1):204-18, as well as in Handbook of TherapeuticAntibodies, Wiley, 2014, Chapter 6, Antibody Affinity (pages 115-140).

In some embodiments there is provided a method of preparing apharmaceutical composition comprising providing an antibody preparedaccording to a method above, (i.e. for producing an antibody by affinitymaturation) and co-formulating the antibody with at least one or morepharmaceutically acceptable excipients. The antibody used in thepreparation of the pharmaceutical composition can be an affinity maturedvariant of aCD38-a-309. The pharmaceutical compositions produced by suchmethods can be used in the methods of treatment of the present inventionas described herein for the other anti-CD38 Antibody Agents.

Antibodies and/or antigen-binding fragments thereof as described herein(e.g., a CD38 Modulating Antibody Agent that may include one or moreaCD38-a-309 amino acid sequence elements such aCD38-a-309-HCDR3 oraCD38-a-309-HCDR123, and/or that may compete with aCD38-a-309 forbinding to human CD38 and non-human primate CD38 for example Cynomolgusmonkey CD38, etc.) may be provided in any of a variety of formats. Forexample, in some embodiments an appropriate format may be or comprise amonoclonal antibody, a domain antibody, a nanobody, a Fab fragment, aF(ab′)2 fragment, a single chain variable fragment (scFv), a scFv-Fcfragment, a single chain antibody (scAb), an aptamer, or a nanobody. Insome embodiments, an antibody or antigen-binding fragment thereof (andparticularly a monoclonal antibody), may be a rabbit, mouse, chimeric,humanized or fully human antibody or antigen-binding fragment thereof.In some embodiments, a provided antibody or antigen-binding fragmentthereof may be of an IgG, IgA, IgE, or IgM isotype (preferably humanones), as it can be most appropriate for a given use. In someembodiments, a provided antibody or antigen-binding fragment thereof isan IgG isotype, more particularly an IgG1, IgG2, IgG3, or IgG4 isotype(preferably human IgG1). In some embodiments, a provided antibody orantigen-binding fragment thereof (e.g., is provided as part of amulti-specific binding agent such as, for example, when it is desirableto associate further binding and/or functional moieties to a CD38Modulating Antibody Agent such as a aCD38-a-309 amino acid sequence, theisolated antibody or antigen-binding can be comprised in a bispecificantibody, a multispecific antibody, or other multi-specific format thatmay be available in the art.

In some embodiments, a provided CD38 Modulating Antibody Agent comprisesa CD38-binding entity (e.g., an anti-CD38 antibody or antigen-bindingfragment thereof) and a conjugated payload such as a therapeutic ordiagnostic agent. In many such embodiments, the agent is consideredand/or referred to as an “immunoconjugate”. Examples of technologies andcompounds that can be used for generating specific immunoconjugates suchas antibody-drug are disclosed in the literature (Beck A et al., 2017)and described as applicable to several known anti-CD38 antibodies(WO2016166304).

In some embodiments, the present invention provides aCD38-a-309 aminoacid sequences that identify provided antibodies or antigen-bindingfragments thereof. In some embodiments, such sequences identify providedantibodies or antigen-binding fragments thereof that bind an epitope inthe extracellular domain of human CD38, and optionally also acorresponding epitope of Cynomolgus monkey and/or murine CD38, either asisolated proteins or on the surface of cells expressing CD38 (such asimmune cells or cell lines, e.g. Raji cells).

The invention also provides CD38 Modulating Antibody Agents binding thesame (or similar) epitope as bound by the CD38 Modulating AntibodyAgents of the invention. For example, in one embodiment there isprovided an antibody that binds the same (or similar) epitope asaCD38-a-309 (or variants thereof)).

In some embodiments, the present invention provides procedures forscreening and/or characterizing antibodies or antigen-binding fragmentsthereof that comprise a aCD38-a-309 amino acid sequences and/or thatpresent binding features comparable to antibodies or antigen-bindingfragments thereof comprising one or more aCD38-a-309 amino acid sequenceelements (e.g. including aCD38-a-309-HCDR3 amino acid sequence and/orcompeting with aCD38-a-309) that allow binding to human CD38extracellular domain as isolated protein and on the surface of cellsexpressing human CD38, competing for the same epitope.

Furthermore, the present invention also provides procedures forscreening antibodies or antigen-binding fragments thereof that presentfunctional features comparable to antibodies or antigen-bindingfragments thereof comprising one or more aCD38-a-309 amino acid sequenceelements, such features being cell activating and cytotoxic activities,and acting as CD38 Modulating Antibody Agents. At these scopes, thecandidate antibodies can be tested in the assays that are described inthe Examples (see FIG. 1) or other assays that are is known in the artfor establishing the presence of any of such features, but possibly allof them when evaluated in in vitro/ex vivo assays, cell-based assays,and/or animal models.

In some embodiments, the present invention provides nucleic acidmolecules encoding an isolated antibody or antigen-binding fragmentthereof that comprises a CD38 Modulating Antibody Agent such as aaCD38-a-309 amino acid sequence. In some embodiments, such providednucleic acid molecules may contain codon-optimized nucleic acidsequences, and/or may be included in expression cassettes withinappropriate nucleic acid vectors for the expression in host cells suchas, for example, bacterial, yeast, insect, piscine, murine, simian, orhuman cells.

In some embodiments, the present invention provides host cellscomprising heterologous nucleic acid molecules (e.g. DNA vectors) thatexpress a provided CD38 Modulating Antibody Agent (e.g., an antibody orantigen-binding fragment thereof) having one or more properties, e.g.,as described herein, of a CD38 Modulating Antibody Agent (e.g.,comprising a aCD38-a-309 amino acid sequence). In some embodiments, thepresent disclosure provides methods of preparing a CD38 ModulatingAntibody Agent (e.g., an antibody or antigen-binding fragment thereof)having one or more properties, e.g., as described herein, of a CD38Modulating Antibody Agent (e.g. comprising a aCD38-a-309 amino acidsequence). In some embodiments, such methods may comprise culturing ahost cell that comprises nucleic acids (e.g., heterologous nucleic acidsthat may comprise and/or be delivered to the host cell via vectors). Insome embodiments, such a host cell (and/or the heterologous nucleic acidsequences) is/are arranged and constructed so that the CD38 ModulatingAntibody Agent (e.g., the antibody or antigen-binding fragment thereof)is secreted from the host cell (e.g., so that it can be isolated fromcell culture supernatants), and/or exposed on the cell surface (forinstance, if such aCD38-a-309 amino acid sequences and sequence elementsare intended to be used in the context of, or together with, such cells,as in artificial T cell receptors grafting the specificity of amonoclonal antibody onto T cells).

In one aspect of the invention, there is provided an anti-CD38 antibodyor antigen-binding fragment thereof, wherein the antibody or fragmentthereof:

-   -   exhibits ADCC activity against CD38+ target cells;    -   exhibits CDC against CD38+ target cells;    -   exhibits direct killing against CD38+ target cells; and    -   increases T cell proliferation in CD4+ and/or CD8+ cells by a        greater amount than for daratumumab under the same or        substantially the same conditions.

In such embodiments, the anti-CD38 antibody or antigen-binding fragmentthereof may be, or may be derived from, for example aCD38-a-309.

In some embodiments, the anti-CD38 antibody or antigen-binding fragmentthereof increases T cell proliferation in CD4+ and/or CD8+ cells by atleast 15% as compared to untreated cells. In some embodiments T cellproliferation increases by at least about 20% as compared to untreatedcells. T cell proliferation may be measured as provided in the Examples,for example as determined at an antibody concentration of 10 ug/ml after72 hours incubation and in the presence of 0.5 μg/ml anti-CD3 antibody.

The anti-CD38 antibody or antigen-binding fragment thereof inducesimmune cell effector cell activation, in particular the antibody inducesT cell activation. In some embodiments, the anti-CD38 antibody orantigen-binding fragment thereof induces T cell activation at a greateramount as compared to daratumumab under the same or substantially thesame conditions.

In some embodiments, the anti-CD38 antibody or antigen-binding fragmentthereof may induce the secretion of cytokines, selected from the groupconsisting of IL-2, TNFa, IFNy and IL-10.

The anti-CD38 antibody or antigen-binding fragment thereof according toclaim 25, wherein the antibody induces the secretion of cytokines,selected from the group consisting of IL-2, TNFa, IFNy and IL-10 at agreater amount than is induced by daratumumab under the same orsubstantially the same conditions. In some embodiments, the anti-CD38antibody or antigen-binding fragment thereof increases the secretion ofIL-2, TNFα, and IL-10 as compared to daratumumab, e.g. aCD38-a-309.Cytokine secretion may be measured as provided in the Examples, forexample as determined at an antibody concentration of 10 ug/ml after 72hours incubation.

In some embodiments T cell activation is determined by measuring NFATsignalling in luc_reporter Jurkat cells. In some embodiments, the NFATsignalling of the anti-CD38 antibody or antigen-binding fragmentthereof, as measured in luc_reporter Jurkat cells is at least about 10%higher than that of daratumumab measured under the same or substantiallythe same conditions. In some embodiments, the NFAT signalling is atleast about 15%, at least about 20%, or at least about 30% higher thanNFAT signalling of daratumumab measured under the same or substantiallythe same conditions.

In a NFAT luc_reporter assay in Jurkat cells, NFAT signalling can bemeasured in the presence of soluble CD3 monoclonal antibody in relativeluminescence units (RLU). The CD3 monoclonal antibody may be at aconcentration of 1 ug/ml and the Jurkat cells may be stimulated with theantiCD38 antibody at a concentration of from about 5 μg/ml to about 40μg/ml (for example 10 μg/ml). Using such an an assay, NFAT signallingmay be at least about 15%, at least about 20%, or at least about 30%higher than NFAT signalling of daratumumab measured under the same orsubstantially the same conditions, when the RLU of CD3 only stimulationis used as a baseline.

CDC activity may be determined using an antibody concentration of up toabout 10 ug/mL. However, maximum lysis may be reached at lowerconcentrations. In some embodiments CDC activity is determined bymeasuring the maximum percentage cell lysis of CD38+ cells, e.g. CD38+Daudi cells in the presence of 10% complement.

In some embodiments, the antibody or fragment thereof causes more than75% lysis of CD38+ expressing cells by CDC. In some embodiments, theantibody or fragment thereof causes more than about 80%, more than about85%, more than about 90% or more than about 95% lysis of CD38+expressing cells by CDC. The percentage lysis can be measured in Daudicells in the presence of 10% complement, at a concentration of about 10ug/ml. In some embodiments, the anti-CD38 antibody or antigen-bindingfragment induces CDC with an EC50 value of more than 0.15 μg/mL againstDaudi cells in the presence of 10% complement. In some embodiments, theanti-CD38 antibody or antigen-binding fragment induces CDC with an EC50value of more than 0.15 μg/mL against Daudi cells in the presence of 10%complement and causes more than 75% lysis of CD38+ expressing cells byCDC.

Antibody-dependent cell-mediated cytotoxicity (ADCC) activity of theanti-CD38 antibodies or antigen-binding fragments thereof may bedetermined in vitro using an assay as described in the Examples, e.g.using CD38+ Daudi cells as the target cell and human PBMC cells aseffector cells, wherein the ratio of target cells to effector cells isbetween 50 to 1 to 25 to 1.

In some embodiments, the anti-CD38 antibody thereof exhibitsantibody-dependant cellular phagocytosis (ADCP) against CD38 expressingcells. ADCP activity may be determined by a reporter cell assaymeasuring FcgRIIa engagement in Jurkat cells as the effector cellsexpressing FcgRIIa. The effector cells also express NFAT-inducedluciferase. The target cell in the assay may be a CD38 expressing Rajicell. NFAT signalling can be measured to determine the activity.

In some embodiments, the anti-CD38 antibody thereof exhibits directkilling of CD38-expressing cells by induction of apoptosis. Directkilling activity may be determined in vitro by using an assay asdescribed in the Examples, e.g. using Daudi cells as target cells andusing the anti-CD38 antibody at increasing concentration up to 10 μg/mL,with or without a secondary (anti-human IgG) antibody or antibodyfragment.

In some embodiments, the anti-CD38 antibody or antigen-binding fragmentthereof has an inhibitory effect on CD38 NADase activity. The inhibitoryeffect on the CD38 NADase activity can be measured as in the assays ofthe Examples, e.g. by measuring the conversion of E-NAD+ into 5′-eAMP inJurkat cells. In some embodiments, the inhibitory effect of theanti-CD38 antibody or antigen-binding fragment thereof on CD38 NADaseactivity is at least 10% lower compared to the CD38 NADase activity inthe presence of an IgG non-binding control antibody as measured by theconversion of E-NAD+ into 5′eAMP in Jurkat cells. In some embodiments,the inhibitory effect can be at least at least about 15% lower comparedto the CD38 NADase activity in Jurkat cells in the presence of an IgGnon-binding control activity.

In some embodiments, the anti-CD38 antibody or antigen-binding fragmentthereof reduces the CD38 NADase activity to no less than about 40% ofthe CD38 NADase activity in the presence of an IgG non-binding controlantibody as measured by the conversion of E-NAD+ into 5′eAMP in Jurkatcells. In some embodiments, the antibody or antigen-binding fragmentthereof reduces the CD38 NADase activity to no less than about 50%, tono less than about 60%, to no less than about 70%, to no less than about75%, to no less than about 80% of the CD38 NADase activity in thepresence of an IgG non-binding control antibody. In some embodiments theantibody may reduce the CD38 NADase activity to between about 40% to90%, between about 50%-90%, between about 60% to 90%, between about 70%to 90%, or to between about 75% to 90% of the CD38 NADase activity inthe presence of an IgG non-binding control antibody. This means that inthe presence of the anti-CD38 antibody or antigen-binding fragmentthereof, CD38 NADase activity is still present in the Jurkat cells,however at a reduced amount as compared to in the presence of an IgGnon-binding control antibody.

In some embodiments, the anti-CD38 antibody or antigen-binding fragmentthereof has an inhibitory effect on CD38 NADase activity, whiledaratumumab has a stimulatory effect on CD38 NADase activity whenmeasured substantially in the same conditions, e.g. when measured inJurkat cells at a concentration of 10 ug/ml.

In some embodiments, the anti-CD38 antibody or antigen-binding fragmentthereof has an inhibitory effect on CD38 cyclase activity. Theinhibitory effect on the CD38 cyclase activity can be measured as in theassays of the Examples, e.g. by measuring the conversion of E-NAD+ into5′eAMP in Jurkat cells. In some embodiments, the inhibitory effect ofthe anti-CD38 antibody or antigen-binding fragment thereof on CD38cyclase activity is at least about 10% lower compared to the CD38cyclase activity in the presence of an IgG non-binding control antibodyas measured by the conversion of E-NAD+ into 5′eAMP in Jurkat cells. Insome embodiments, the inhibitory effect can be at least at least about15% lower, at least about 20% lower, at least about 30% lower, at leastabout 40% lower, at least about 50% lower or at least about 60% lowercompared to the CD38 cyclase activity in the presence of an IgGnon-binding control activity.

In some embodiments, the anti-CD38 antibody or antigen-binding fragmentreduces the CD38 cyclase activity to no less than about 40% of the CD38cyclase activity in the presence of an IgG non-binding control antibodyas measured by the conversion of E-NAD+ into 5′eAMP in Jurkat cells. Insome embodiments, the antibody or antigen-binding fragment thereofreduces the CD38 cyclase activity to no less than about 45%, or to noless than about 50% of the CD38 cyclase activity in the presence of anIgG non-binding control antibody. In some embodiments the antibody mayreduce the CD38 cyclase activity to between about 40%-95%, between about40%-80%, between about 40% to 70%, between about 40% to 60%, or tobetween about 45% to 55% of the CD38 cyclase activity in the presence ofan IgG non-binding control antibody. This means that in the presence ofthe anti-CD38 antibody or antigen-binding fragment thereof, CD38 cyclaseactivity is still present in the Jurkat cells, however at a reducedamount as compared to in the presence of an IgG non-binding controlantibody.

In some embodiments, the inhibitory effect of the anti-CD38 antibody orantigen-binding fragment CD38 on CD38 cyclase activity is less than anyinhibitory effect of daratumumab on CD38 cyclase activity when measuredin the same or substantially the same conditions, e.g. when measured inJurkat cells at a concentration of 10 ug/ml.

The present invention also includes variants or derivates of theantibody aCD38-a-309. Variant or derivative antibodies or antigenbinding fragments thereof may share the same functional profile (i.e.pharmacological properties) as for the antibody from which they arederived. Similarly, the present invention includes antibodies or antigenbinding fragments that compete for binding to CD38 with aCD38-a-309 (orvariants thereof). Such competing antibodies may have the samefunctional profile (i.e. pharmacological properties) as aCD38-a-309.

In some embodiments the antibody or antigen-binding fragment thereof (orvariants of the same) may be afucosylated. It is well known thatantibody glycosylation may have impact on the activity, pharmacokineticsand pharmacodynamics of antibodies (e.g., monoclonal antibodies,recombinant antibodies, and/or antibodies that are otherwise engineeredor isolated) and Fc-fusion proteins and specific technology may beexploited to obtain an antibody with the desired glycosylation profile(Liu L, 2015). Effector functions supporting the cytotoxicity of anantibody for use in accordance with the present invention (e.g., ananti-CD38 antibody as described herein, including for example anantibody which may be or be described as a CD38 Modulating AntibodyAgent) can be enhanced using methods to decrease antibody fucosylationlevels. Antibodies comprising specific aCD38-a-309 sequence elementspresenting such properties can be generated, for example, by expressinga aCD38-a-309 sequence using technologies for genetically engineeringcell lines which may produce antibodies with absent or reducedfucosylation capacity, some of them commercially available such asPotelligent (Lonza) GlyMAXX (ProBiogen), or by manipulating themanufacturing process, for example by controlling osmolarity and/orusing enzyme inhibitors , see also for example the methods described inEP2480671.

In some embodiments, the present invention provides compositions (e.g.pharmaceutical compositions) comprising a provided antibody or anantigen-binding fragment thereof having desirable properties asdescribed herein (e.g., as described for antibodies that are hereintermed CD38 Modulating Antibody Agents, specifically including, forexample, aCD38-a-309 antibodies or antigen-binding fragments thereof,and variants thereof). In some embodiments, such a provided compositionsare intended for and/or are used in a medical use, such as atherapeutic, diagnostic, or prophylactic use. In some embodiments, aprovided such composition can further comprise a pharmaceuticallyacceptable carrier or excipient and/or may be for use in the treatmentof cancer. In some embodiments, a pharmaceutical composition may beformulated with one or more carrier, excipients, salts, bufferingagents, etc., as is known in the art. Those of skill in the art will beaware of and readily able to utilize a variety of formulationtechnologies, including as may be particularly desirable and/or usefulfor a given method and/or site of administration, for instance forparenteral (e.g. subcutaneous, intramuscular, or intravenous injection),mucosal, intratumoral, peritumoral, oral, or topical administration. Inmany embodiments, provided pharmaceutical compositions, comprising aCD38 Modulating Antibody Agent as described herein (e.g., an anti-CD38antibody or antigen binding portion thereof), are formulated forparenteral delivery (e.g., by injection and/or infusion). In someembodiments, such a provided pharmaceutical composition may be provided,for example, in a pre-loaded syringe or vial format. In someembodiments, such a provided pharmaceutical composition may be providedand/or utilized, for example, in dry (e.g., lyophilized) form;alternatively, in some embodiments, such a provided pharmaceuticalcomposition may be provided and/or utilized in a liquid form (e.g., as asolution, suspension, dispersion, emulsion, etc.), in a gel form, etc.

In some embodiments, the present invention provides uses of CD38Modulating Antibody Agents (e.g., anti-CD38 antibodies orantigen-binding fragments thereof) as described herein (e.g. comprisinga aCD38-a-309 amino acid sequence element), and/or of a compositioncomprising them, in treatment of and/or in the manufacture of amedicament for treatment of, a cancer, such as a B cell malignancy, alymphoma, (Hodgkins Lymphoma, non-Hodgkins lymphoma, chroniclymphocytic, leukemia, acute lymphoblastic leukemia, myelomas), amyeloproliferative disorders, a solid tumor (such as a breast carcinoma,a squamous cell carcinoma, a colon cancer, a head and neck cancer, alung cancer, a genitourinary cancer, a rectal cancer, a gastric cancer,sarcoma, melanoma, an esophageal cancer, liver cancer, testicularcancer, cervical cancer, mastocytoma, hemangioma, eye cancer, laryngealcancer, mouth cancer, mesothelioma, skin cancer, rectal cancer, throatcancer, bladder cancer, breast cancer, uterine cancer, prostate cancer,lung cancer, pancreatic cancer, renal cancer, gastric cancer, non-smallcell lung cancer, and ovarian cancer). The cancer can be also defined onthe basis of presence of specific tumor-relevant markers and antigenssuch as CD20, HER2, PD-1, PD-L1, SLAM7F, CD47, CD137, CD134, TIM3, CD25,GITR, EGFR, etc. or a cancer that has been identified as having abiomarker referred to as microsatellite instability-high (MSI-H) ormismatch repair deficient (dMMR). Furthermore, such conditions may alsobe considered when defining pre-cancerous, non-invasive states of theabove cancers, such as cancer in-situ, smouldering myeloma, monoclonalgammopathy of undetermined significance, cervical intra-epithelialneoplasia, MALTomas/GALTomes and various lymphoproliferative disorders.Preferably in some embodiments the subject being treated has a solidtumor. In one embodiment the subject has a heamatological cancer. Insome embodiments the subject has a CD38 positive tumor.

Thus, in some embodiments, the present invention provides methods oftreating cancer in a subject, comprising administering to the subject aneffective amount of a composition comprising a provided CD38 ModulatingAntibody Agent (e.g., anti-CD38 antibodies or antigen-binding fragmentsthereof) as described herein (e.g. comprising aCD38-a-309 amino acidsequences). In some embodiments, provided methods may further compriseadministering, simultaneously or sequentially in any order, at least oneadditional agent or therapy to the subject (i.e., so that the subjectreceives a combination therapy). In some embodiments, such an at leastone additional agent or therapy can be or comprise an anticancer drug(e.g., a chemotherapeutic agent), radiotherapy (by applying irradiationexternally to the body or by administering radio-conjugated compounds),an anti-tumor antigen or marker antibody (the antigen or marker beingfor example CD4, CD25, CA125, PSMA, c-MET, VEGF, CD137, VEGFR2, CD20,HER2, HER3, SLAMF7, CD326, CAIX, CD40, CD47, or EGF receptor), acheckpoint inhibitor or an immunomodulating antibody (for example anantibody targeting PD-1. PD-L1, TIM3, CD25, GITR, CD134, CD134L, CD137L,CD80, CD86, B7-H3, B7-H4, B7RP1, LAG3, ICOS, TIM3, GAL9, CD28, AP2M1,SHP-2, OX-40 etc.), a vaccine, an adjuvant, standard-of-use protocol,one or more other compounds targeting cancer cells or stimulating animmune response against cancer cells, or any combination thereof. Incertain particular embodiments, when such at least one additional agentor therapy is or comprises an antibody, the format of and/or the antigentargeted by such antibody can be chosen among those listed in theliterature and possibly adapted to a given cancer (Sliwkowski M &Mellman I, 2013; Redman J M et al., 2015; Kijanka M et al., 2015).

Still further, the present invention provides a variety of kits orarticles of manufacture containing a provided CD38 Modulating AntibodyAgent (e.g., anti-CD38 antibody or antigen-binding fragment thereof) asdescribed herein (e.g. comprising aCD38-a-309 amino acid sequences) orrelated compositions that allow the administration, storage, or otheruse of such an isolated antibody or antigen-binding fragment. In someembodiments, a provided kit comprises a vessel, syringe, a vial, orother container comprising such compositions, optionally together withone or more articles of manufactures, diluents, reagents, solid phases,and/or instructions for the correct use of the kit.

In some embodiments, identification, characterization, and/or validationof particular CD38 Modulating Antibody Agent (e.g., anti-CD38 antibodyor antigen-binding fragment thereof) as described herein (e.g.comprising aCD38-a-309 amino acid sequences) for a particular use, suchas a medical use, and in particular for treating cancer, can beperformed by using one or more assays or systems as described herein. Insome embodiments, such identification, characterization, and/orvalidation may involve analysis of activity in one or more cell-basedassays, for example using different experimental set-ups and/or a panelof selected (e.g., cancer-derived cell lines). In some embodiments,particularly given the proposed immunological mechanism associatedcertain desirable CD38 Modulating Antibody Agents as described hereinactivities, desirable identification, characterization, and/orvalidation can involve collection of relevant data generated in animalmodels wherein cancers are induced or wherein cancer cells are implantedas a xenograft or as a syngeneic/allogeneic cancer-derived cells.Alternatively or additionally, in some embodiments, animal models may beutilized that involve transfer of human cells such as PBMC (i.e.humanized PBMC mouse models) or CD34+ hematopoietic stem cells (i.e.CD34+ humanized mice) to allow evaluating activity of the CD38Modulating Antibody Agents on human immune cells within a model system.

In some embodiments, relevant sequences of CD38 Modulating AntibodyAgents (e.g., anti-CD38 antibody or antigen-binding fragments thereof)as described herein (e.g. comprising aCD38-a-309 amino acid sequences orotherwise including structural and/or functional characteristics of anagent described herein as a CD38 Modulating Antibody Agent) can becloned into and/or expressed in context of an antibody frame that ismore appropriate or desirable for pharmaceutical and/or technicalreasons. For example, such sequences (possibly as codon-optimized VH andVL coding sequences) can be cloned together with human IgG1 constantregions (hIgG1) and expressed using an appropriate antibody expressionvectors and cell line (such as a CHO-derived cell line, e.g. CHO-S). Insome particular embodiments, expression and secretion of providedantibody sequences in human IgG1 format antibodies can be analyzed aftertransfection in reduced conditions in cell lysates and in non-reducedconditions in supernatants that will be later used to purify theantibody (by affinity chromatography, gel filtration, and/or otherappropriate technique). Binding and/or other functional properties ofprovided anti-CD38 antibody sequences, in human IgG1 format (e.g., CD38Modulating Antibody Agents-hIgG1) can be analysed, for example by usingone or more assays described in Examples below. For instance, suchhIgG1-format provided antibodies can be evaluated for binding to humanand cynomolgus PBMC, e.g., using flow cytometry. Alternatively oradditionally, binding to specific immune cell populations can beassessed, for example using flow cytometry that may employ one or morespecific markers for specific immune cell populations, like CD3, CD45,CD56 and CD159 (NKG2A) for NK cells, CD14 (for monocytes), CD19 (for Bcells), and/or CD4/CD8 (for T cells).

Moreover, the effect of one or more CD38 Modulating Antibody Agents(e.g., anti-CD38 antibody or antigen-binding fragments thereof) asdescribed herein (e.g. comprising aCD38-a-309 amino acid sequences (orvariants thereof) or otherwise including structural and/or functionalcharacteristics of an agent described herein as a CD38 ModulatingAntibody Agent—such as a CD38 Modulating Antibody Agent-hIgG1) on humanprimary tumor cells and/or immune cells isolated from human healthydonors and/or patients can be assessed. In order to investigatepotential effects on individual immune cell populations in more detail,such CD38 Modulating Antibody Agents can be used to treat PBMC and/orcells isolated from tumors (and/or organs such as lymph nodes) and/orpurified human CD8 and CD4 T cells, Treg cells, MDSC cells, dendriticcells, macrophages and monocytes, neutrophils, NK cells and other celltypes. Potential read outs comprise cytokine release, tumor cellkilling, cell proliferation, and/or activation, apoptosis,antigen-specific and/or allogenic responses, or any combination thereof.Alternatively or additionally, mice or non human primates can be treatedand cellular status can be followed using flow cytometry or afterisolation of various organs and/or cells from the animals.

Alternatively or additionally, one or more properties of CD38 ModulatingAntibody Agents (e.g., anti-CD38 antibody or antigen-binding fragmentsthereof) as described herein (e.g. comprising aCD38-a-309 amino acidsequences (or variants thereof) or otherwise including structural and/orfunctional characteristics of an agent described herein as a CD38Modulating Antibody Agent—such as a CD38 Modulating AntibodyAgent-hIgG1) may be evaluated, alone or in combination, by studying theeffects of such CD38 Modulating Antibody Agents on CD38 expressing cells(e.g. NK cells or T-cells); CD38 enzymatic activity, CD38 induced Ca²⁺levels and protein phosphorylation, CD38 shedding and/orinternalization, CD38-induced activation of intra-cellular pathways(e.g. NFKB pathway), and/or interaction with CD31 and other receptorproteins (e.g., CD16, TCR, BCR, etc.). Involvement of the latterprocesses in the CD38 downstream activity can also be evaluated usingspecific inhibitors of these processes. These cellular effects can thenbe followed in vivo when aCD38 Modulating Antibody Agent-hIgG1antibodies are administered to cynomolgus monkeys.

In order to gain further insights into the molecular interactionsbetween a provided CD38 Modulating Antibody Agent and human CD38, thecrystal structure of the CD38 Modulating Antibody Agent (e.g., to giveone specific example, a aCD38-a-309-hIgG1 antibody) and human CD38protein can be determined. Solubility and/or stability of provided CD38Modulating Antibody Agents (specifically including, for example,aCD38-a-309-hIgG1 antibodies) can be assessed through solubilitystudies, accelerated stress studies, freeze thaw studies and formalstability studies. Aggregation of the antibodies can be followed byvisual inspection, size exclusion chromatography and dynamic lightscattering and OD280/320 absorbance.

The present invention includes variants or derivates of the antibodyaCD38-a-309. Variant or derivative antibodies or antigen bindingfragments thereof may share the same functional profile (i.e.pharmacological properties) as for the antibody from which they arederived. Similarly, the present invention includes antibodies or antigenbinding fragments that compete for binding to aCD38-a-309 (or variantsthereof). Such competing antibodies may have the same functional profile(i.e. pharmacological properties) as the aCD38-a-309.

BRIEF DESCRIPTION OF DRAWINGS

FIG. 1: flowchart summarizing the screening procedure for identifyingaCD38-a-309 as an agonistic anti-CD38 antibody having one or moreproperties according to the present invention, in particular thosedescribed herein as characteristic of CD38 Modulating Antibody Agents ofparticular interest: pharmaceutically relevant targeted cell killing(e.g. as measured in ADCC, ADCP, and CDC assays), effects on immunecells (such as Treg, CD8 and CD4 T cells, NK cells, dendritic cells,MDSC, macrophages, and/or monocytes, for measuring properties such ascell viability and/or proliferation, cytokine secretion, and/oractivation markers), effects on CD38 enzymatic activities orCD38-mediated signaling, effects on cancer cells expressing (or not)CD38, combinations with other drugs (e.g. antibodies targeting a tumorantigen or other anticancer drugs) and/or antibody sequence and format,for identifying stability issues related to aggregation-prone sequences,presence of glycosylation sites or free Cysteines in variable domainand/or effects (e.g. within a human IgG1 frame, as Fabs, single domainantibodies, bi/multispecific antibodies, or within non-antibodyscaffolds).

FIG. 2: relevant protein sequences aCD38-a-309 protein sequences. EachCDR for the heavy (aCD38-a-309-HCDR1 (SEQ ID NO: 1), aCD38-a-309-HCDR2(SEQ ID NO: 2), and aCD38-a-309-HCDR3 (SEQ ID NO: 3)) and the light(aCD38-a-309-LCDR1 (SEQ ID NO: 5), aCD38-a-309-LCDR2 (SEQ ID NO: 6), andaCD38-a-309-LCDR3 (SEQ ID NO: 7) chain is indicated separately and,underlined, within the frame sequence of the heavy and light chainantibody as initially identified by the screening procedure(aCD38-a-309-HCDR123 (SEQ ID NO: 4) and aCD38-a-309-LCDR123(SEQ ID NO:8), respectively).

FIG. 3: characterization of aCD38-a-309 binding to CD38 expressed oncell lines of human origin at increasing antibody concentration andcomparing with either human IgG1 isotype control, or Daratumumab (DARA).

FIG. 4: functional characterization of aCD38-a-309 compared toDaratumumab (DARA) or negative control antibodies (anti-human CD3 orhuman IgG1 isotype), in cell-based models independently from theadministration of any further tumor targeting antibody. (A) aCD38-a-309increases the percentage of TCR-mediated CD4 and CD8 T cellproliferation, as indicated in each graph. (DARA was tested at 10-5-2.5μg/ml; anti-CD3 is tested at 0.5 μg/ml and IgG1 and aCD38-a-309 wereused at 10 μg/ml). (B) aCD38-a-309 increases the secretion of selectedcytokines, especially IL-2, TNFa and IL10, by TCR-activated CD4/CD8 Tcells (similar pattern in 3 out of 3 donors tested).

FIG. 5: functional characterization of aCD38-a-309 compared to DARA withrespect to cytotoxicity. (A) Both aCD38-a-309 and DARA induce killing byantibody-dependent cell-mediated cytotoxicity (ADCC) and (B)complement-dependent cytotoxicity (CDC, particularly significant forDARA, as described in the literature). (C) Moreover, both aCD38-a-309and DARA induce direct cell killing with and without crosslinking.

FIG. 6: functional characterization of aCD38-a-309 compared toDaratumumab (DARA) and control antibody (human IgG1 isotype) withrespect to enzymatic activity. Tested is the inhibition or activation ofCD38 cyclase or NADase (hydrolase) activity by aCD38-309 and controlantibodies. (A) aCD38-a-309 and DARA inhibit cyclase activity (N=2experiments). (B) In Daudi cells, which express very high levels ofCD38, it was observed that both aCD38-a-309 and DARA do not affect theNADase (NAD+ hydrolase) activity (N=2 experiments). However, laterexperiments conducted in Jurkat cells, which express much less CD38 thanDaudi cells, showed that aCD28-a-309 does inhibit NADase activity andDARA stimulated NADase activity (FIG. 10), indicating that the effectcan be cell-type specific and be dependent on the CD38 proteinexpression levels.

FIG. 7: shows the binding of anti-CD38 antibodies as compared todaratumumab to recombinant human CD38 his tagged measured by biolayerinterferometry on the Octet Red 96 instrument. 4.2 nM of rhCD38-his wasloaded to the Ni NTA biosensor followed by 7 nM of antibody and then letthem to dissociate in Kinetics Buffer.

FIG. 8: shows the binding aCD38-a-309 (FIG. 8A) as compared todaratumumab (FIG. 8B) to recombinant human CD38 his tagged measured bybiolayer interferometry on the Octet Red 96 instrument. 4.2nM ofrhCD38-his was loaded to the Ni NTA biosensor followed by varyingconcentrations of antibody (as shown in the Figures) and then let themto dissociate in Kinetics Buffer.

FIG. 9: functional characterization of aCD38-a-309 compared to DARA withrespect to cytotoxicity. aCD38-a-309 shows ADCP activity as measuredusing a NFAT reporter assay in Jurkat cells

FIG. 10: functional characterization of aCD38-a-309 compared toDaratumumab (DARA) and control antibody (human IgG1 isotype) withrespect to enzymatic activity. Tested is the inhibition or activation ofCD38 cyclase or NADase (hydrolase) activity by aCD38-309 and controlantibodies in Jurkat cell based models. (A) aCD38-a-309 and DARA inhibitcyclase activity. (B) aCD38-a-309 slightly inhibited NADase (NAD+hydrolase) activity (about 15% inhibition) while DARA stimulated NADaseactivity (about 30% stimulation).

FIG. 11: Characterisation of aCD38-a-309 (administered at 10 mg/kg) withrespect to animal survival in an in vivo solid tumour cancer model basedover the indicated number of days. The treatment with aCD38-a-309increased animal survival not only when compared to negative control butalso when compared to daratumumab (DARA).

FIG. 12: characterization of aCD38-a-309 (administered at 10 mg/kg) withrespect to animal survival in an in vivo solid tumor cancer model basedon the intra-venous administration of Ramos cells over the indicatednumber of days, as compared to daratumumab. Impact of treatment on micesurvival is evaluated.

FIG. 13: characterization of aCD38-a-309 (administered at 10 mg/kg) withrespect to animal survival in an in vivo tumor cancer model based on theintra-venous administration of Raji cells over the indicated number ofdays, as compared to daratumumab. Impact of treatment on mice survivalis evaluated.

FIG. 14: shows the CDC of aCD38-a-309 compared to Dara and human IgG1isotype control antibody in Raji cells with 10% complement. In thisexperiment, aCD38-a-309 exhibited higher CDC than Dara.

FIG. 15: SPR based analysis of purified aCD38-a-309 antibody (IgG1) torhCD38 his tagged on the Biacore 2000.

DETAILED DESCRIPTION OF CERTAIN EMBODIMENTS

Below are provided certain definitions of terms, technical means, andembodiments used herein, many or most of which confirm commonunderstanding of those skille12d in the art.

Administration: As used herein, the term “administration” refers to theadministration of a composition to a subject. Administration to ananimal subject (e.g., to a human) may be by any appropriate route. Forexample, in some embodiments, administration may be bronchial (includingby bronchial instillation), buccal, enteral, intra-arterial,intra-dermal, intra-gastric, intra-medullary, intra-muscular,intra-nasal, intra-peritoneal, intra-thecal, intra-venous,intra-ventricular, within a specific organ or tissue (e. g.intra-hepatic, intra-tumoral, peri-tumoral, etc.), mucosal, nasal, oral,rectal, subcutaneous, sublingual, topical, tracheal (including byintra-tracheal instillation), transdermal, vaginal and vitreal. Theadministration may involve intermittent dosing. Alternatively,administration may involve continuous dosing (e.g., perfusion) for atleast a selected period of time. As is known in the art, antibodytherapy is commonly administered parenterally, e.g. by intravenous,subcutaneous, or intratumoral injection (e.g., particularly when highdoses within a tumor are desired).

Agent: The term “agent” as used herein may refer to a compound or entityof any chemical class including, for example, polypeptides, nucleicacids, saccharides, small molecules, metals, or combinations thereof.Specific embodiments of agents that may be utilized in accordance withthe present invention include small molecules, drugs, hormones,antibodies, antibody fragments, aptamers, nucleic acids (e.g., siRNAs,shRNAs, antisense oligonucleotides, ribozymes), peptides, peptidemimetics, etc. An agent may be or comprise a polymer.

Antibody: As used herein, the term “antibody” refers to a polypeptidethat includes canonical immunoglobulin sequence elements sufficient toconfer specific binding to a particular target antigen, such as CD38,human CD38 in particular, and human CD38 extracellular domain. As isknown in the art, intact antibodies as produced in nature areapproximately 150 kD tetrameric agents comprised of two identical heavychain polypeptides (about 50 kD each) and two identical light chainpolypeptides (about 25 kD each) that associate with each other into whatis commonly referred to as a “Y-shaped” structure. Each heavy chain iscomprised of at least four domains (each about 110 amino acids long), anamino-terminal variable (VH) domain (located at the tips of the Ystructure), followed by three constant domains: CH1, CH2, and thecarboxy-terminal CH3 (located at the base of the Y's stem). A shortregion, known as the “switch”, connects the heavy chain variable andconstant regions. The “hinge” connects CH2 and CH3 domains to the restof the antibody. Two disulfide bonds in this hinge region connect thetwo heavy chain polypeptides to one another in an intact antibody. Eachlight chain is comprised of two domains—an amino-terminal variable (VL)domain, followed by a carboxy-terminal constant (CL) domain, separatedfrom one another by another “switch”. Intact antibody tetramers arecomprised of two heavy chain-light chain dimers in which the heavy andlight chains are linked to one another by a single disulfide bond; twoother disulfide bonds connect the heavy chain hinge regions to oneanother, so that the dimers are connected to one another and thetetramer is formed. Naturally produced antibodies are also glycosylated,typically on the CH2 domain, and each domain has a structurecharacterized by an “immunoglobulin fold” formed from two beta sheets(e.g., 3-, 4-, or 5-stranded sheets) packed against each other in acompressed antiparallel beta barrel. Each variable domain contains threehypervariable loops known as “complement determining regions” (CDR1,CDR2, and CDR3; as understood in the art, for example determinedaccording to Kabat numbering scheme) and four somewhat invariant“framework” regions (FR1, FR2, FR3, and FR4). When natural antibodiesfold, the FR regions form the beta sheets that provide the structuralframework for the domains, and the CDR loop regions from both the heavyand light chains are brought together in three-dimensional space so thatthey create a single hypervariable antigen-binding site located at thetip of the Y structure. The Fc region of naturally-occurring antibodiesbinds to elements of the complement system, and also to receptors oneffector cells, including for example effector cells that mediatecytotoxicity. As is known in the art, affinity and/or other bindingattributes of Fc regions for Fc receptors can be modulated throughglycosylation or other modification that can improve the developabilityof the antibody (Jarasch A et al., 2015).

In some embodiments, antibodies produced and/or utilized in accordancewith the present invention include glycosylated Fc domains, including Fcdomains with modified or engineered such glycosylation. For purposes ofthe present invention, in certain embodiments, any polypeptide orcomplex of polypeptides that includes sufficient immunoglobulin domainsequences as found in natural antibodies can be referred to and/or usedas an “antibody”, whether such polypeptide is naturally produced (e.g.,generated by an organism reacting to an antigen), or produced byrecombinant engineering, chemical synthesis, or other artificial systemor methodology. In some embodiments, an antibody is polyclonal oroligoclonal, that is generated as a panel of antibodies, each associatedto a single antibody sequence and binding a more or less distinctepitopes within an antigen (such as different epitopes within human CD38extracellular domain that are associated to different referenceanti-CD38 antibodies).

Polyclonal or oligoclonal antibodies can be provided in a singlepreparation for medical uses as described in the literature (Kearns J Det al., 2015). In some embodiments, an antibody is monoclonal. In someembodiments, an antibody has constant region sequences that arecharacteristic of mouse, rabbit, primate, or human antibodies. In someembodiments, antibody sequence elements are humanized, primatized,chimeric, etc., as is known in the art. Moreover, the term “antibody” asused herein, can refer in appropriate embodiments (unless otherwisestated or clear from context) to any of the art-known or developedconstructs or formats for utilizing antibody structural and functionalfeatures in alternative presentation, for instance as antigen-bindingfragments as defined below. For example, an antibody utilized inaccordance with the present invention is in a format selected from, butnot limited to, intact IgG, IgE and IgM, bi- or multi-specificantibodies (e.g., Zybodies®, etc.), single chain variable domains(scFv), polypeptide-Fc fusions, Fabs, cameloid antibodies, heavy-chainshark antibody (IgNAR), masked antibodies (e.g., Probodies®), or fusionproteins with polypeptides that allow expression and exposure on thecell surface (as scFv within constructs for obtaining artificial T cellreceptors that are used to graft the specificity of a monoclonalantibody onto a T cell). A masked antibody can comprise a blocking or“mask” peptide that specifically binds to the antigen binding surface ofthe antibody and interferes with the antibody's antigen binding. Themask peptide is linked to the antibody by a cleavable linker (e.g. by aprotease). Selective cleavage of the linker in the desired environment,e.g. in the tumour environment, allows the masking/blocking peptide todissociate, enabling antigen binding to occur in the tumour, and therebylimiting potential toxicity issues. In some embodiments, an antibody maylack a covalent modification (e.g., attachment of a glycan) that itwould have if produced naturally. Alternatively, an antibody may containa covalent modification (e.g., attachment of a glycan, a payload [e.g.,a detectable moiety, a therapeutic moiety, a catalytic moiety, etc.], orother pendant group [e.g., poly-ethylene glycol, etc.])

Antigen: The term “antigen”, as used herein, refers to an agent thatelicits an immune response and/or that binds to a T cell receptor (e.g.,when presented by an MHC molecule) and/or B cell receptor. An antigenthat elicits a humoral response involve the production ofantigen-specific antibodies or, as shown in the Examples for CD38extracellular domain, can be used for screening antibody libraries andidentifying candidate antibody sequences to be further characterized.

Antigen-binding Fragment: As used herein, the term “Antigen-bindingFragment” encompasses agents that include or comprise one or moreportions of an antibody as described herein sufficient to confer on theantigen-binding fragment and ability to specifically bind to the Antigentargeted by the antibody. For example, in some embodiments, the termencompasses any polypeptide or polypeptide complex that includesimmunoglobulin structural elements sufficient to confer specificbinding. Exemplary antigen-binding fragments include, but are notlimited to Small Modular ImmunoPharmaceuticals (“SMIPs™”), single chainantibodies, cameloid antibodies, single domain antibodies (e.g., sharksingle domain antibodies), single chain or Tandem diabodies (TandAb®),VHHs, Anticalins®, Nanobodies®, minibodies, BiTE®s, ankyrin repeatproteins or DARPINs®, Avimers®, a DART, a TCR-like antibody, Adnectins®,Affilins®, Trans-bodies®, Affibodies®, a TrimerX®, MicroProteins,Centyrins®, CoVX bodies, BiCyclic peptides, Kunitz domain derivedantibody constructs, or any other antibody fragments so long as theyexhibit the desired biological activity. In some embodiments, the termencompasses other protein structures such as stapled peptides,antibody-like binding peptidomimetics, antibody-like binding scaffoldproteins, monobodies, and/or other non-antibody proteins scaffold, forexample as reviewed in the literature (Vazquez-Lombardi R et al., 2015).In some embodiments, an antigen-binding fragment is or comprises apolypeptide whose amino acid sequence includes one or more structuralelements recognized by those skilled in the art as a complementaritydetermining region (CDR). In some embodiments an antigen-bindingfragment is or comprises a polypeptide whose amino acid sequenceincludes at least one reference CDR (e.g., at least one heavy chain CDRand/or at least one light chain CDR) that is substantially identical toone found in an anti-CD38 antibody as described herein (e.g., in anaCD38-a-309 amino acid sequence element), and in particular at least oneheavy chain CDR, such as an HCDR3 (e.g., an aCD38-a-309-HCDR3 sequence).In some embodiments an antigen-binding fragment is or comprises apolypeptide whose amino acid sequence includes at least one CDR (e.g.,at least one heavy chain CDR and/or at least one light chain CDR) thatis either identical in sequence or contains a small number (e.g., 1, 2,3, or 4) more amino acid alterations (e.g., substitutions, additions, ordeletions; in many cases, substitutions) relative to such a referenceCDR, while maintaining binding to the target of the antibody (e.g.,aCD38-a-309) from which the reference CDR was derived. In someembodiments, an antigen-binding fragment is or comprises a polypeptideor complex thereof that includes all three CDRs (or, in someembodiments, sequences substantially identical thereto) from a heavy orlight chain of a reference antibody (e.g., from aCD38-a-309); in someembodiments, an antigen-binding fragment is or comprises a polypeptideor complex thereof that includes all six CDRs (or, in some embodiments,sequences substantially identical thereto) from a reference antibody(e.g., from aCD38-a-309). In some embodiments, an antigen-bindingfragment is or comprises a polypeptide or complex thereof that includesthe heavy and/or light chain variable domains (or, in some embodiments,sequences substantially identical thereto) of a reference antibody(e.g., of aCD38-a-309). In some embodiments, the term “antigen-bindingfragment” encompasses non-peptide and non-protein structures, such asnucleic acid aptamers, for example, RNA aptamers and DNA aptamers. Anaptamer is an oligonucleotide (e.g., DNA, RNA, or an analog orderivative thereof) that binds to a particular target, such as apolypeptide. Aptamers are short synthetic single-strandedoligonucleotides that specifically bind to various molecular targetssuch as small molecules, proteins, nucleic acids, and even cells andtissues. These small nucleic acid molecules can form secondary andtertiary structures capable of specifically binding proteins or othercellular targets, and are essentially a chemical equivalent ofantibodies. Aptamers are highly specific, relatively small in size, andnon-immunogenic. Aptamers are generally selected from a biopanningmethod known as SELEX (Systematic Evolution of Ligands by Exponentialenrichment) (See for example Ellington et al. Nature. 1990; 346(6287):818-822; Tuerk et al., Science. 1990; 249(4968):505-510; Ni et al., CurrMed Che 2011; 18(27):4206-14). Methods of generating an apatmer for anygiven target are well known in the art. Peptide aptamers includingaffimers are also encompassed. An affimer is a small, highly stableprotein engineered to display peptide loops which provide a highaffinity binding surface for a specific target protein. It is a proteinof low molecular weight, 12-14 kDa, derived from the cysteine proteaseinhibitor family of cystatins. Affimer proteins are composed of ascaffold, which is a stable protein based on the cystatin protein fold.They display two peptide loops and an N-terminal sequence that can berandomized to bind different target proteins with high affinity andspecificity similar to antibodies. Stabilization of the peptide upon theprotein scaffold constrains the possible conformations which the peptidemay take, thus increasing the binding affinity and specificity comparedto libraries of free peptides.

Percent (%) sequence identity: Percent (%) “sequence identity” betweentwo sequences can be determined using those methods known in the art.Sequence identity with respect to a peptide, polypeptide or antibodysequence can be defined as the percentage of amino acid residues in acandidate sequence that are identical with the amino acid residues inthe specific peptide or polypeptide sequence, after aligning thesequences and introducing gaps, if necessary, to achieve the maximumpercent sequence identity, and not considering any conservativesubstitutions as part of the sequence identity. Alignment for purposesof determining percent amino acid sequence identity can be achieved invarious ways that are within the skill in the art, for instance, usingpublicly available computer software such as BLAST, BLAST-2, includinggapped BLAST, and BLASTp (for proteins), (Altschul S F et al (1997)), orFASTA, using the default parameters.

Biological Sample. As used herein, the terms “biological sample” or”“sample” typically refers to a sample obtained or derived from abiological source (e.g., a tissue or organism or cell culture) ofinterest, as described herein. A source of interest may be an organism,such as an animal or human. The biological sample may comprisebiological tissue or fluid.

Cancer: The terms “cancer”, “malignancy”, “neoplasm”, “tumor”, “tumour”,and “carcinoma”, are used interchangeably herein to refer to cells thatexhibit relatively abnormal, uncontrolled, and/or autonomous growth, sothat they exhibit an aberrant growth phenotype characterized by asignificant loss of control of cell proliferation. In general, cells ofinterest for detection or treatment in the present application includeprecancerous (e.g., benign), malignant, pre-metastatic, metastatic, andnon-metastatic cells. The teachings of the present disclosure may berelevant to any and all cancers. To give but a few, non-limitingexamples, in some embodiments, teachings of the present disclosure areapplied to one or more cancers such as, for example, hematopoieticcancers including leukemias, lymphomas (Hodgkins and non-Hodgkins),myelomas and myeloproliferative disorders; sarcomas, melanomas,adenomas, carcinomas of solid tissue, squamous cell carcinomas of themouth, throat, larynx, and lung, liver cancer, genitourinary cancerssuch as prostate, cervical, bladder, uterine, and endometrial cancer andrenal cell carcinomas, bone cancer, pancreatic cancer, skin cancer,cutaneous or intraocular melanoma, cancer of the endocrine system,cancer of the thyroid gland, cancer of the parathyroid gland, head andneck cancers, breast cancer, gastro-intestinal cancers and nervoussystem cancers, benign lesions such as papillomas, and the like. Theantibodies of the invention can be used in the treatment of CD38+expressing tumours

CD38 Modulating Antibody Agent The term “CD38 Modulating Antibody Agent”is used herein to refer to those CD38 Modulating Antibody Agents (e.g.,anti-CD38 antibodies) that demonstrate particular properties asdescribed herein. In many embodiments, desirable CD38 ModulatingAntibody Agents as described herein are characterized in that theystimulate immune effector cells and/or modify immune cell function andare cytotoxic towards or induce phagocytosis of CD38 expressing cells(e.g. expressing high levels of CD38) such as immune suppressive cellsor tumour cells (e.g., in each case, that express CD38 on theirsurfaces). In some embodiments, a CD38 Modulating Antibody Agent ischaracterized by an activity (e.g., level and/or type) reasonablycomparable to that of aCD38-a-309 with respect to immune cells (e.g.,when contacted with immune cells, and particularly with immune cellsthat express CD38) and tumour cells. In some embodiments, a relevantactivity is or comprises ADCP, ADCC, CDC, direct killing, depletion ofcertain CD38-expressing cells (e.g., high-expressing cells), effectorimmune cell activation, promotion of T cell, B cell or NK cellexpansion, modulation of immune cells activity (e.g. repolarization ofsuppressive macrophages into inflammatory macrophages), skewing of Tcell repertoire, etc., and combinations thereof. In some embodiments,CD38 Modulating Antibody Agents are entities or moieties whose presenceor level correlates with level and/or activity of CD38, and/or with oneor more features or results characteristic of CD38 activity. In someembodiments, an increased level and/or activity is assessed ordetermined relative to that observed under otherwise comparableconditions in absence of the entity(ies) or moiety(ies). Alternativelyor additionally, in some embodiments, an increased level and/or activityis comparable to or greater than that observed under comparableconditions when a reference CD38 Modulating Antibody Agent (e.g., anappropriate reference anti-CD38 antibody, which in many embodiments is aCD38 agonist antibody, such as IB4) is present. In many embodiments, aCD38 Modulating Antibody Agent for use in accordance with the presentdisclosure is or comprises an entity or moiety that binds, directly orindirectly, to CD38, typically to its extracellular domain. In someembodiments, a CD38 Modulating Antibody Agent is, comprises, or competesfor binding to CD38 with an anti-CD38 antibody as exemplified herein, anantigen-binding fragment (e.g., comprising one or more CDRs, all heavychain CDRs, all light chain CDRs, all CDRs, a heavy chain variableregion, a light chain variable region, or both heavy and light chainvariable regions) thereof, an affinity matured variant thereof (or anantigen-binding fragment thereof), or any alternative format (e.g.,chimeric, humanized, multispecific, alternate isotype, etc.) of any ofthe foregoing. Alternatively or additionally, in some embodiments, aCD38 Modulating Antibody Agent as described hereinmay be characterizedby one or more features that may be features that are advantageous forscreening, manufacturing, (pre-)clinical testing, and/or for identifyingrelevant epitope within human CD38), and/or for formulation,administration, and/or efficacy in particular contexts (e.g., for cancertherapy), as disclosed herein.

Combination Therapy: As used herein, the term “combination therapy”refers to those situations in which a subject is simultaneously exposedto two or more therapeutic regimens (e.g., two or more therapeuticagents). In some embodiments, two or more agents may be administeredsimultaneously. Alternatively, such agents may be administeredsequentially; otherwise, such agents are administered in overlappingdosing regimens.

Comparable: As used herein, the term “comparable” refers to two or moreagents, entities, situations, effects, sets of conditions, etc., thatmay not be identical to one another but that are sufficiently similar topermit comparison (e.g., by level and/or activity) there between so thatconclusions may reasonably be drawn based on differences or similaritiesobserved. Such comparable sets of conditions, effects, circumstances,individuals, or populations are characterized by a plurality ofsubstantially identical features and one or a small number of variedfeatures. Those of ordinary skill in the art will understand, incontext, what degree of identity is required in any given circumstancefor two or more such agents, entities, situations, sets of conditions,effects, or populations, etc. to be considered comparable.

Comprising: A composition or method described herein as “comprising” oneor more named elements or steps is open-ended, meaning that the namedelements or steps are essential, but other elements or steps may beadded within the scope of the composition or method. It is alsounderstood that any composition or method described as “comprising” (orwhich “comprises”) one or more named elements or steps also describesthe corresponding, more limited composition or method “consistingessentially of” (or which “consists essentially of”) the same namedelements or steps, meaning that the composition or method includes thenamed essential elements or steps and may also include additionalelements or steps that do not materially affect the basic and novelcharacteristic(s) of the composition or method.

Daratumumab: As used herein, the term “daratumumab” includes an antibodyhaving, VH and VL sequences as published in WO2006/099875 and being ahuman IgG1 monoclonal antibody. For example having variable heavy andlight chain sequences comprising the respective sequences as providedbelow:

(SEQ ID NO: 14) Heavy Chain:EVQLLESGGGLVQPGGSLRLSCAVSGFTFNSFAMSWVRQAPGKGLEWVSAISGSGGGTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYFCAKDKILWFGEPVFDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSP GK (SEQ ID NO: 15)Light Chain EIVLTQSPATLSLSPGERATLSCRASQSVSSYLAWYQQKPGQAPRLLIYDASNRATGIPARFSGSGSGTDFTLTISSLEPEDFAVYYCQQRSNWPPTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQG LSSPVTKSFNRGEC.

Dosage Form: As used herein, the term “dosage form” refers to aphysically discrete unit of an active agent (e.g., a therapeutic ordiagnostic agent) for administration to a subject. Each unit contains apredetermined quantity of active agent. In some embodiments, suchquantity is a unit dosage amount (or a whole fraction thereof)appropriate for administration in accordance with a dosing regimen thathas been determined to correlate with a desired or beneficial outcomewhen administered to a relevant population (i.e., with a therapeuticdosing regimen). Those of ordinary skill in the art appreciate that thetotal amount of a therapeutic composition or agent administered to aparticular subject is determined by one or more attending physicians andmay involve administration of multiple dosage forms.

Dosing Regimen: As used herein, the term “dosing regimen” refers to aset of unit doses (typically more than one) that are administeredindividually to a subject, typically separated by periods of time. Insome embodiments, a given therapeutic agent has a recommended dosingregimen, which may involve one or more doses. In some embodiments, adosing regimen comprises a plurality of doses each of which areseparated from one another by a time period of the same length.Alternatively, a dosing regimen comprises a plurality of doses and atleast two different time periods separating individual doses. In someembodiments, all doses within a dosing regimen are of the same unit doseamount. Alternatively, different doses within a dosing regimen are ofdifferent amounts. In some embodiments, a dosing regimen comprises afirst dose in a first dose amount, followed by one or more additionaldoses in a second dose amount different from the first dose amount. Adosing regimen may comprise a first dose in a first dose amount,followed by one or more additional doses in a second dose amount same asthe first dose amount. In some embodiments, a dosing regimen iscorrelated with a desired or beneficial outcome when administered acrossa relevant population (i.e., is a therapeutic dosing regimen).

Epitope: As used herein, the term “epitope” refers to a portion of anantigen that is bound by an antibody or antigen-binding fragment. Insome embodiments, where the antigen is a polypeptide, an epitope isconformational in that it is comprised of portions of an antigen thatare not covalently contiguous in the antigen but that are near to oneanother in three-dimensional space when the antigen is in a relevantconformation. For example, for CD38, conformational epitopes are thosecomprised of amino acid residues that are not contiguous in CD38extracellular domain; linear epitopes are those comprised of amino acidresidues that are contiguous in CD38 extracellular domain. In someembodiments, epitopes utilized in accordance with the present inventionare provided by means of reference to those bound by CD38 ModulatingAntibody Agents provided herein (e.g., by aCD38-a-309). Means fordetermining the exact sequence and/or particularly amino acid residuesof the epitope for aCD38-a-309 are known in the literature and in theExamples, including competition with peptides, from antigen sequences,binding to CD38 sequence from different species, truncated, and/ormutagenized (e.g. by alanine scanning or other site-directedmutagenesis), phage display-based screening, or (co-)crystallographytechniques.

Patient: As used herein, the term “patient” or “subject” refers to anyorganism to which a provided composition is or may be administered,e.g., for experimental, diagnostic, prophyl actic, cosmetic, and/ortherapeutic purposes. Typical patients include animals (e.g., mammalssuch as mice, rats, rabbits, non-human primates, and/or humans). In someembodiments, a patient is a human. In some embodiments, a patient issuffering from or susceptible to one or more disorders or conditions. Apatient may display one or more symptoms of a disorder or condition, ormay have been diagnosed with one or more disorders or conditions (suchas cancer, or presence of one or more tumors). In some embodiments, thepatient is receiving or has received certain therapy to diagnose and/orto treat such disease, disorder, or condition.

Pharmaceutically Acceptable: As used herein, the term “pharmaceuticallyacceptable” applied to the carrier, diluent, or excipient used toformulate a composition as disclosed herein means that the carrier,diluent, or excipient must be compatible with the other ingredients ofthe composition and not deleterious to the recipient thereof.

Pharmaceutical Composition: As used herein, the term “pharmaceuticalcomposition” refers to a composition in which an active agent isformulated together with one or more pharmaceutically acceptablecarriers. In some embodiments, active agent is present in unit doseamount appropriate for administration in a therapeutic regimen thatshows a statistically significant probability of achieving apredetermined therapeutic effect when administered to a relevantpopulation. A pharmaceutical compositions may be formulated foradministration in solid or liquid form, including those adapted for thefollowing: oral administration, for example, drenches (aqueous ornon-aqueous solutions or suspensions), tablets, e.g., those targeted forbuccal, sublingual, and systemic absorption, boluses, powders, granules,pastes for application to the tongue; parenteral administration, forexample, by subcutaneous, intramuscular, intravenous, intratumoral, orepidural injection as a sterile solution or suspension, orsustained-release formulation; topical application, for example, as acream, ointment, or a controlled-release patch or spray applied to skin,lungs, or oral cavity; intravaginally, intrarectally, sublingually,ocularly, transdermally, nasally, pulmonary, and to other mucosalsurfaces.

Solid Tumor. As used herein, the term “solid tumor” refers to anabnormal mass of tissue that usually does not contain cysts or liquidareas. Solid tumors may be benign or malignant. Different types of solidtumors are named for the type of cells that form them. Examples of solidtumors are sarcomas (including cancers arising from transformed cells ofmesenchymal origin in tissues such as cancellous bone, cartilage, fat,muscle, vascular, hematopoietic, or fibrous connective tissues),carcinomas (including tumors arising from epithelial cells), melanomas,lymphomas, mesothelioma, neuroblastoma, retinoblastoma, etc. Cancersinvolving solid tumors include, without limitations, brain cancer, lungcancer, stomach cancer, duodenal cancer, esophagus cancer, breastcancer, colon and rectal cancer, renal cancer, bladder cancer, kidneycancer, pancreatic cancer, prostate cancer, ovarian cancer, melanoma,mouth cancer, sarcoma, eye cancer, thyroid cancer, urethral cancer,vaginal cancer, neck cancer, lymphoma, and the like.

Therapeutically Effective Amount: As used herein, the term“therapeutically effective amount” means an amount (e.g., of an agent orof a pharmaceutical composition) that is sufficient, when administeredto a population suffering from or susceptible to a disease and/orcondition in accordance with a therapeutic dosing regimen, to treat suchdisease and/or condition. A therapeutically effective amount is one thatreduces the incidence and/or severity of, stabilizes, and/or delaysonset of, one or more symptoms of the disease, disorder, and/orcondition. Those of ordinary skill in the art will appreciate that a“therapeutically effective amount” does not in fact require successfultreatment be achieved in a particular subject.

Treatment: As used herein, the term “treatment” (also “treat” or“treating”) refers to any administration of a substance (e.g., aprovided CD38 Modulating Antibody Agent, as exemplified by aCD38-a-309,or any other agent) that partially or completely alleviates,ameliorates, relives, inhibits, delays onset of, reduces severity of,and/or reduces incidence of one or more symptoms. In some embodiments,treatment may involve the direct administration of a CD38 ModulatingAntibody Agent such as aCD38-a-309 (for example, as an injectable,aqueous composition, optionally comprising a pharmaceutically acceptablecarrier, excipient and/or adjuvant, for use for intravenous,intratumoral or peritumoral injection) or the administration using aregimen comprising obtaining cells from the subject (e.g. from theblood, a tissue, or a tumor, with or without a selection on the basis ofpresence, or absence, of the expression of a marker), contacting saidcells with a CD38 Modulating Antibody Agent such as aCD38-a-309 ex vivo,and administering such cells to the subject (with or without a selectionon the basis of presence, or absence, of the expression of a marker).

Dosing and Administration. Pharmaceutical compositions comprising a CD38Modulating Antibody Agent as described herein (e.g., an anti-CD38 orantigen-binding fragment thereof, for example comprising theaCD38-a-309-HCDR3 amino acid sequence) for use in accordance with thepresent invention may be prepared for storage and/or delivery using anyof a variety of techniques and/or technologies known and/or available tothose skilled in the art. In some embodiments, a provided CD38Modulating Antibody Agent is administered according to a dosing regimenapproved by a regulatory authority such as the United States Food andDrug Administration (FDA) and/or the European Medicines Agency (EMEA),e.g., for the relevant indication. In some embodiments, a provided CD38Modulating Antibody Agent is administered in combination with one ormore other agents or therapies, which may themselves be administeredaccording to a dosing regimen approved by a regulatory authority such asthe United States Food and Drug Administration (FDA) and/or the EuropeanMedicines Agency (EMEA), e.g., for the relevant indication. In someembodiments however, use of a provided CD38 Modulating Antibody Agentmay permit reduced dosing (e.g., lower amount of active in one or moredoses, smaller number of doses, and/or reduced frequency of doses) of anapproved agent or therapy used in combination with the CD38 ModulatingAntibody Agent therapy. In some embodiments, dosing and/oradministration may be adapted to other drugs that also administered, thepatient status, and/or the format of CD38 Modulating Antibody Agent(e.g. modified as an immunoconjugate, a nanobody, or a bispecificantibody).

Moreover, in some embodiments, it may be desirable to tailor dosingregimens, and particularly to design sequential dosing regimens, basedon timing and/or threshold expression levels of CD38, whether forparticular cell types, particular tumors or types thereof, or particularpatient populations (e.g., carrying genetic markers). In some suchembodiments, therapeutic dosing regimens may be combined with oradjusted in light of detection methods that assess expression of one ormore inducible markers or other criteria prior to and/or during therapy.

In some embodiments, dosing and administration according to the presentinvention utilizes active agent having a desired degree of puritycombined with one or more physiologically acceptable carriers,excipients or stabilizers in any or variety of forms. These include, forexample, liquid, semi-solid and solid dosage forms, such as liquidsolutions (e.g., injectable and infusible solutions), dispersions orsuspensions, tablets, pills, powders, liposomes and suppositories. Apreferred form may depend on the intended mode of administration and/ortherapeutic application, typically in the form of injectable orinfusible solutions, such as compositions similar to those used fortreating of human subjects with antibodies.

In some embodiments, ingredient(s) can be prepared with carriers thatprotect the agent(s) against rapid release and/or degradation, such as acontrolled release formulation, including implants, transdermal patches,and microencapsulated delivery systems. Biodegradable, biocompatiblepolymers can be used, such as polyanhydrides, polyglycolic acid,polyorthoesters, and polylactic acid. In general, each active agent isformulated, dosed, and administered in therapeutically effective amountusing pharmaceutical compositions and dosing regimens that areconsistently with good medical practice and appropriate for the relevantagent(s) (e.g., for agents such as antibodies). Pharmaceuticalcompositions containing active agents can be administered by anyappropriate method known in the art, including, without limitation,oral, mucosal, by-inhalation, topical, buccal, nasal, rectal, orparenteral (e.g. intravenous, infusion, intratumoral, intranodal,subcutaneous, intraperitoneal, intramuscular, intradermal, transdermal,or other kinds of administration involving physical breaching of atissue of a subject and administration of the pharmaceutical compositionthrough such breach).

In some embodiments, a dosing regimen for a particular active agent mayinvolve intermittent or continuous (e.g., by perfusion or slow releasesystem) administration, for example to achieve a particular desiredpharmacokinetic profile or other pattern of exposure in one or moretissues or fluids of interest in the subject. In some embodiments,different agents administered in combination may be administered viadifferent routes of delivery and/or according to different schedules.Alternatively, or additionally, in some embodiments, one or more dosesof a first active agent is administered substantially simultaneouslywith, and in some embodiments via a common route and/or as part of asingle composition with, one or more other active agents.

Factors to be considered when optimizing routes and/or dosing schedulefor a given therapeutic regimen may include, for example, the particularcancer being treated (e.g., type, stage, location, etc.), the clinicalcondition of a subject (e.g., age, overall health, weight, etc.), thesite of delivery of the agent, the nature of the agent (e.g. an antibodyor other protein-based compound), the mode and/or route ofadministration of the agent, the presence or absence of combinationtherapy, and other factors known to medical practitioners.

Those skilled in the art will appreciate, for example, that a specificroute of delivery may impact dose amount and/or required dose amount mayimpact route of delivery. For example, where particularly highconcentrations of an agent within a particular site or location (e.g.,within a tissue or organ) are of interest, focused delivery (e.g.,intratumoral delivery) may be desired and/or useful. In someembodiments, one or more features of a particular pharmaceuticalcomposition and/or of a utilized dosing regimen may be modified overtime (e.g., increasing or decreasing amount of active in any individualdose, increasing or decreasing time intervals between doses, etc.), forexample in order to optimize a desired therapeutic effect or response(e.g., a therapeutic or biological response that is related to thefunctional features of a CD38 Modulating Antibody Agent as describedherein). In general, type, amount, and frequency of dosing of activeagents in accordance with the present invention in governed by safetyand efficacy requirements that apply when relevant agent(s) is/areadministered to a mammal, preferably a human. In general, such featuresof dosing are selected to provide a particular, and typicallydetectable, therapeutic response as compared with what is observedabsent therapy. In context of the present invention, an exemplarydesirable therapeutic response may involve, but is not limited to,inhibition of and/or decreased tumor growth, tumor size, metastasis, oneor more of the symptoms and side effects that are associated with thetumor, as well as increased apoptosis of cancer cells, therapeuticallyrelevant decrease or increase of one or more cell marker or circulatingmarkers and the like. Such criteria can be readily assessed by any of avariety of immunological, cytological, and other methods that aredisclosed in the literature. For example, the therapeutically effectiveamount of CD38 Modulating Antibody Agent, alone or in combination with afurther agent, can be determined as being sufficient to enhance killingof cancer cells as described in the Examples.

A therapeutically effective amount of a CD38 Modulating Antibody Agentas active agent or composition comprising such agent can be readilydetermined using techniques available in the art including, for example,considering one or more factors such as the disease or condition beingtreated, the stage of the disease, the age and health and physicalcondition of the mammal being treated, the severity of the disease, theparticular compound being administered, and the like.

In some embodiments, therapeutically effective amount is an effectivedose (and/or a unit dose) of an active agent that may be at least about0.01 mg/kg body weight, at least about 0.05 μg/kg body weight; at leastabout 0.1 μg/kg body weight, at least about 1 mg/kg body weight, atleast about 5 μg/kg body weight, at least about 10 mg/kg body weight, ormore (e.g. 100 μg/kg body weight). It will be understood by one of skillin the art that in some embodiments such guidelines may be adjusted forthe molecular weight of the active agent. The dosage may also be variedfor route of administration, the cycle of treatment, or consequently todose escalation protocol that can be used to determine the maximumtolerated dose and dose limiting toxicity (if any) in connection to theadministration of the isolated antibody or antigen-binding fragmentthereof comprising the aCD38-a-309-HCDR3 amino acid sequence atincreasing doses.

Therapeutic compositions typically should be sterile and stable underthe conditions of manufacture and storage. The composition can beformulated as a solution, microemulsion, dispersion, liposome, or otherordered structure suitable to high drug concentration. Sterileinjectable solutions can be prepared by incorporating the antibody inthe required amount in an appropriate solvent with one or a combinationof ingredients enumerated above, followed by filtered sterilization.Generally, dispersions are prepared by incorporating the active compoundinto a sterile vehicle that contains a basic dispersion medium and otherrequired ingredients from those enumerated above. In the case of powdersfor preparing sterile injectable solutions, the preferred methods ofpreparation are vacuum drying and freeze drying that yields a powder ofthe active ingredient plus any additional desired ingredient from apreviously sterile filtered solution. The proper fluidity of a solutioncan be maintained, for example, by using a coating, by the maintenanceof the required particle size in the case of dispersion and by the useof surfactants. Prolonged absorption of injectable compositions can bebrought about by including in the composition an agent that delaysabsorption, for example, monostearate salts and gelatin.

The formulation of each agent should desirably be sterile, as can beaccomplished by filtration through sterile filtration membranes, andthen packaged, or sold in a form suitable for bolus administration orfor continuous administration. Injectable formulations may be prepared,packaged, or sold in unit dosage form, such as in ampules or in multidose containers containing a preservative. Formulations for parenteraladministration include, but are not limited to, suspensions, solutions,emulsions in oily or aqueous vehicles, pastes, and implantablesustained-release or biodegradable formulations as discussed herein.Sterile injectable formulations may be prepared using a non-toxicparenterally acceptable diluent or solvent, such as water or 1,3butanediol. Other parentally-administrable formulations which are usefulinclude those which comprise the active ingredient in microcrystallineform, in a liposomal preparation, or as a component of biodegradablepolymer systems. Compositions for sustained release or implantation maycomprise pharmaceutically acceptable polymeric or hydrophobic materialssuch as an emulsion, an ion exchange resin, a sparingly soluble polymeror salt.

Each pharmaceutical composition for use in accordance with the presentinvention may include pharmaceutically acceptable dispersing agents,wetting agents, suspending agents, isotonic agents, coatings,antibacterial and antifungal agents, carriers, excipients, salts, orstabilizers are non-toxic to the subjects at the dosages andconcentrations employed. A non-exhaustive list of such additionalpharmaceutically acceptable compounds includes buffers such asphosphate, citrate, and other organic acids; antioxidants includingascorbic acid and methionine; salts containing pharmacologicallyacceptable anions (such as acetate, benzoate, bicarbonate, bisulfate,isothionate, lactate, lactobionate, laurate, malate, maleate,salicylate, stearate, subacetate, succinate, tannate, tartrate,teoclate, tosylate, thiethiodode, and valerate salts); preservatives(such as octadecyidimethylbenzyl ammonium chloride; hexamethoniumchloride; benzalkonium chloride, benzethonium chloride; sodium chloride;phenol, butyl or benzyl alcohol; alkyl parabens such as methyl or propylparaben; catechol; resorcinol; cyclohexanol; 3-pentanol; and m-cresol);low molecular weight (less than about 10 residues) polypeptides;proteins, such as serum albumin; hydrophilic polymers such aspolyvinylpyrrolidone; amino acids such as glycine, glutamine,asparagine, glutamic acid, histidine, arginine, or lysine;monosaccharides, disaccharides, and other carbohydrates includingglucose, mannose, or dextrins; chelating agents such as EDTA; sugarssuch as sucrose, mannitol, trehalose or sorbitol; salt-formingcounter-ions such as sodium; metal complexes (e.g., Zn-proteincomplexes); and/or non-ionic surfactants such as TWEEN™, PLURONICS™, orpolyethylene glycol (PEG).

In some embodiments, where two or more active agents are utilized inaccordance with the present invention, such agents can be administeredsimultaneously or sequentially. In some embodiments, administration ofone agent is specifically timed relative to administration of anotheragent. In some embodiments, desired relative dosing regimens for agentsadministered in combination may be assessed or determined empirically,for example using ex vivo, in vivo and/or in vitro models; in someembodiments, such assessment or empirical determination is made in vivo,in a particular patient or patient population (e.g., so that acorrelation is made).

In some embodiments, one or more active agents utilized in practice ofthe present invention is administered according to an intermittentdosing regimen comprising at least two cycles. Where two or more agentsare administered in combination, and each by such an intermittent,cycling, regimen, individual doses of different agents may beinterdigitated with one another. In some embodiments, one or more dosesof the second agent is administered a period of time after a dose of aCD38 Modulating Antibody Agent as described herein. In some embodiments,each dose of the second agent is administered a period of time after adose of CD38 Modulating Antibody Agent as described herein. In someembodiments, a CD38 Modulating Antibody Agent as described herein can bealso administered in regimens that involve not only subsequentadministration by the same route but also by alternating administrationroutes such as by sub-cutaneous (or intramuscular) administration andintra-tumoral administration, within one or more cycles of treatmentsover one, two, four or more weeks, repeating such cycle with the sameregimen (or by extending the interval between administrations),depending of patient responses. Also, in some embodiments, the preciseregimen followed (e.g., number of doses, spacing of doses (e.g.,relative to each other or to another event such as administration ofanother therapy), amount of doses, etc. may be different for one or morecycles as compared with one or more other cycles.

By using any of the routes of administrations, dosages, and/or regimensas described herein, a CD38 Modulating Antibody Agent as describedherein can be identified, characterized, and/or validated, for example,taking into account one or more criteria that are measured in thepatients using biopsies, blood samples, and/or other clinical criteria.In some embodiments, as an alternative or in addition to directevaluation of tumor size and/or metastasis, therapeutic efficacy of aCD38 Modulating Antibody Agent as described herein can be determined inmethods wherein one or more different general criteria are evaluated:direct cytotoxicity on cancer cells (apoptosis and necrosis of cancercells), increase of tumor infiltrating, immune cells (such asCD4-positive and/or CD8-positive tumor infiltrating T cells), increasein immune cells that circulates in blood (total populations or specificsub-populations of lymphocytes, NK cells, monocytes, dendritic cells,macrophages, B cells, etc.), and/or presenting some differentialexpression pre-versus post-treatment only in either responding ornon-responding patients (as determined by RNA sequencing, mass flowcytometry, and/or other mass sequencing approach). Alternatively oradditionally, in some embodiments, such identification,characterization, and/or validation may involve the follow-up atmolecular level by screening the mRNA and/or protein expression of oneor more specific proteins or sets of proteins. In some embodiments, oneor more such techniques may allow identification or relevant informationfor evaluating the response to a CD38 Modulating Antibody Agent asdescribed herein, for example that may be is related to tissuedistribution and/or markers for specific cell populations within (ornearby) the tumor and/or circulating in blood.

Such approaches and immune-biological data may allow determination notonly of one or more efficacy and/or safety parameters orcharacteristics, but in some embodiments, can provide a rationale forchoosing a particular dose, route or dosing regimen, for example thatmay be utilized in one or more clinical trials for a given indication,alone and/or in combination with other drugs, standard-of-careprotocols, or immunotherapies that can provide further therapeuticbenefits. Thus, in a series of further embodiments of the invention, aCD38 Modulating Antibody Agent as described herein is used in a methodof treating a patient suffering from a disease (such as cancer) orpreventing a disease (such as cancer) after determining the combinedpresence (and/or absence) of expression at RNA and/or protein level forone or more genes in cells or tissues of the patient (such as a tumor, ablood sample, or a blood fraction), post- or pre-treatment with such aformulation. Such methods may allow therefore defining a one or morebiomarkers, or a more complex gene expression signature (or cellpopulation distribution) that is associated to the therapeuticallyeffective amount of a desirable CD38 Modulating Antibody Agent, thetherapeutically relevant biomarker(s) that predicts that a subject mayhave an anti-tumor or anti-infective response after the treatment with aCD38 Modulating Antibody Agent as described herein, or thetherapeutically relevant biomarker(s) that predicts that a subject mayrespond to the treatment with a compound after the treatment with a CD38Modulating Antibody Agent.

Alternatively or additionally, in some embodiments, dosing andadministration for a particular CD38 Modulating Antibody Agent asdisclosed herein can be preliminarily established and/or later evaluatedin view of CD38 expression in human cancers and/or other human tissues,for example by gathering data about CD38 distribution in stromal and/orimmune subsets in various cancers, tissues, and/or patients. Such datacan be generated by using common technologies (such as flow cytometry,mass cytometry, immunohistochemistry or mRNA expression libraries)across common cancer types and/or tissues (central nervous system,Esophagus, Stomach, Liver, Colon, Rectum, Lung, Bladder, Heart, Kidney,Thyroid, Pancreas, Uterus, Skin, Breast, Ovary, Prostate and testis) foridentifying relationship between CD38 expression in various immune andnon immune subpopulations and/or its relation with cell infiltratemeasures and/or cancer-relevant markers associated with sub-sets ofcancer cells or immune cells (such as Foxp3 and PD-1/PD-L1). CD38expression can be confined (or not) to immune subsets in tumor tissue(such as in NK cells and other effector or regulatory immune cells), andcorrelations between CD38 expression and immune checkpoint inhibitorscan be determined if being positive, thus suggesting appropriate uses ofCD38 Modulating Antibody Agents in combinations with compounds targetingsuch immune checkpoint inhibitors.

Articles of Manufacture and Kits; In some embodiments of the invention,a CD38 Modulating Antibody Agent as described herein is provided in aseparate article of manufacture. In some embodiments of the invention,an article of manufacture containing a CD38 Modulating Antibody Agent isprovided in or with a container with a label. Suitable containers mayinclude, for example, bottles, vials, syringes, and test tubes. In someembodiments, a container may be formed from any or a variety ofmaterials such as glass or plastic. In some embodiments, a containerholds a composition that is effective for treating a particular disease,disorder, or condition, or stage or type thereof. In some embodiments, acontainer may have a sterile access port (for example the container maybe an intravenous solution bag or a vial having a stopper pierceable bya hypodermic injection needle). For example, in some embodiments,compositions comprising a CD38 Modulating Antibody Agent as describedherein is packaged in clear glass vials with a rubber stopper and analuminium seal. The label on, or associated with, the containerindicates that the composition is used for treating the condition ofchoice.

In some embodiments, an article of manufacture may further comprise aseparate container comprising a pharmaceutically acceptable buffer, suchas phosphate-buffered saline, Ringer's solution and dextrose solutionand/or may further include other materials desirable from a commercialand user standpoint, including other buffers, diluents, filters,needles, syringes, and package inserts with instructions for use. Forexample, in some embodiments, an article of manufacture may allowproviding each or the agent in an intravenous formulation as a sterileaqueous solution containing a total of 2 mg, 5 mg, 10 mg, 20 mg, 50 mg,or more that are formulated, with appropriate diluents and buffers, at afinal concentration of 0.1 mg/ml, 1 mg/ml, 10 mg/ml, or at a higherconcentration.

In some embodiments, a CD38 Modulating Antibody Agent as describedherein can be provided within the kits-of-parts in the form oflyophilized is to be reconstituted with any appropriate aqueous solutionthat provided or not with the kits, or other types of dosage unit usingany compatible pharmaceutical carrier. One or more unit dosage forms ofa CD38 Modulating Antibody Agent may be provided in a pack or dispenserdevice. Such a pack or device may, for example, comprise metal orplastic foil, such as a blister pack. In order to use correctly suchkits-of-parts, it may further comprise buffers, diluents, filters,needles, syringes, and package inserts with instructions for use in thetreatment of cancer.

In some embodiments, instructions that are associated with an article ofmanufacture or the kits as described herein may be in the form of alabel, a leaflet, a publication, a recording, a diagram, or any othermeans that can be used to inform about the correct use and/or monitoringof the possible effects of the agents, formulations, and other materialsin the article of manufacture and/or in the kit. Instructions may beprovided together with the article of manufacture and/or in the kit.

EXAMPLES Example 1 Generation of Antibodies that Bind CD38 In Vitro

Materials & Methods

CD38 antigen preparation. Recombinant, Histidine-tagged extracellulardomain of human, Cynomolgus monkey (Cyno), and murine CD38 proteins werepurchased from Sino Biological Inc. Protein reagent biotinylation wasdone using the EZ-Link Sulfo-NHS-Biotinylation Kit (Thermo Scientific,Cat #21425). The CD38 antigen was concentrated to ˜1mg/mL and bufferexchanged into PBS before addition of 1:7.5 molar ratio biotinylationreagents (EZ-Link Sulfo-NHS-Biotinylation Kit, Thermo Scientific, Cat#21425). The mixture was held at 4° C. overnight prior to another bufferexchange to remove free biotin in the solution. Biotinylation wasconfirmed through Streptavidin sensor binding of the labelled proteins.

Library interrogation and selection methodology for isolation ofanti-CD38 antibodies: Eight naïve human synthetic yeast libraries eachof ˜109 diversity were designed, generated, and propagated forhigh-throughput screening and selection of yeast cell lines expressingmonoclonal antibodies as described previously (Xu Y et al, 2013;WO2009036379; WO2010105256; WO2012009568). Eight parallel selectionswere performed, using the eight naïve libraries for monomeric humanCD38-based selection.

For the first two rounds of selection, a magnetic bead sorting techniqueutilizing the Miltenyi MACs system was performed, essentially asdescribed (Siegel et al., 2004). Briefly, yeast cells (10¹⁰cells/library) were incubated with 3 ml of 100 nM biotinylated monomerichuman CD38 antigen for 15 minutes at room temperature in FACS washbuffer PBS with 0.1% BSA. After washing once with 50 ml ice-cold washbuffer, the cell pellet was re-suspended in 40 mL wash buffer, and 500μl Streptavidin MicroBeads (Miltenyi Biotec, Germany. Cat #130-048-101)were added to the yeast cells and incubated for 15 minutes at 4° C.Next, the yeast cells were pelleted, resuspended in 5 mL wash buffer,and loaded onto a MACS LS column (Miltenyi Biotec, Germany. Cat. No.130-042-401). After the 5 mL was loaded, the column was washed 3 timeswith 3 ml FACS wash buffer. Column was removed from magnetic field,yeast cells were eluted with 5 mL growth media, and then grownovernight.

Subsequent to the two MACS rounds, five rounds of sorting were performedusing flow cytometry (FACS). For the first round of FACS selection,approximately 4×10⁷ yeast cells were pelleted, washed three times withwash buffer, and incubated with 100 nM of each the biotinylatedmonomeric human, murine, and Cyno CD38 antigen for 10 minutes at roomtemperature. Yeast cells were then washed twice and stained with goatanti-human F(ab′)2 kappa-FITC diluted 1:100 (Southern Biotech, USA; Cat.No. 2062-02) and either streptavidin-Alexa Fluor 633 (Life Technologies,USA; Cat. No. S21375) diluted 1:500, or Extravidin-phycoerthyrin(Sigma-Aldrich, USA; Cat. No. E4011) diluted 1:50, secondary reagentsfor 15 minutes at 4° C. After washing twice with ice-cold wash buffer,cell pellets were resuspended in 0.4 mL wash buffer and transferred tostrainer-capped sort tubes. Sorting was performed using a FACS ARIAsorter (BD Biosciences) and sort gates were determined to select onlyCD38 binding. Murine- and Cyno-selected populations from the first FACSround were combined into two pools. These pools were then sorted forhuman CD38 binding to identify cross-reactive binders in the second FACSround to decrease reagent polyspecific binders (Xu Yet al., 2013). Thefourth FACS round consisted predominantly of positive selection using100 nM biotinylated monomeric CD38 as antigen. A sample of the selectedclones were plated and sequenced.

Affinity Maturation of clones identified in naive selections: Heavychains from the fourth FACS sorting selection round outputs were used toprepare light chain diversification libraries used for four additionalselection rounds. The first selection round involved Miltenyi MACs beadsconjugated with either 100 nM biotinylated monomeric human CD38 asantigen or 200 nM biotinylated monomeric murine CD38 as antigen.Subsequent to the MACs bead selections, three rounds of FACS sortingwere performed. The first FACS round involved either human CD38 at 100nM or 10 nM or murine CD38 at 200 nM. In parallel to the second FACSround described above, competition selections were performed with 75-100nM of competitor IgG. After a selection round, a third positive sortwith human CD38 at 1 or 10 nM was done before plating. Individualcolonies from each FACS selection round were picked for sequencing IgG.

IgG and Fab production & purification: Yeast clones were grown tosaturation and then induced for 48 hrs at 30° C. with shaking. Afterinduction, yeast cells were pelleted and the supernatants were harvestedfor purification. IgGs were purified using a Protein A column and elutedwith acetic acid, pH 2.0. Fab fragments were generated by papaindigestion and purified over CaptureSelect IgG-CH1 affinity matrix (LifeTechnologies; Cat. No. 1943200250).

Affinity Measurements of anti-CD38 Antibodies: The affinity for the CD38antibodies was determined by measuring their KD by Forte Bio. Forte Bioaffinity measurements were performed by loading IgGs on-line onto AHQsensors as described (Estep P et al., 2013). Briefly, sensors wereequilibrated off-line in assay buffer for 30 minutes and then monitoredon-line for 60 seconds for baseline establishment. For avid bindingmeasurement, sensors with loaded IgGs were exposed to 200 nM of human,cyno, or murine CD38 for 3 minutes, afterwards they were transferred toassay buffer for 3 minutes for off-rate measurement. Monovalent bindingmeasurements were obtained by loading biotinylated CD38 monomer on SAsensors followed by exposure to 200 nM Fab. Kinetics data were fit usinga 1:1 binding model of data analysis software provided by Forte Bio. TheKd values that were established in this assay for the referenceagonistic anti-CD38 antibodies are the following: for IB4, 0.9×10⁻⁸ Mfor human CD38 and no binding to cynomolgus CD38, for IB4.

Alternatively, the affinity for the anti-human CD38 antibodies wasdetermined by measuring their K_(D) by SPR in a Biacore 2000 using aCM-5 Sensor chip with an ambient experiment temperature of 25° C.Anti-human antibody was initially immobilised across all flow cells inanalysis buffer (pH 7.4, 10 mM HEPES, 150 mM NaCl, 3 mM EDTA, 0.05%Tween 20) to an RU of between 12,000-14,000 over 10 minutes. The ligand(antibody test articles) was sub sequentially loaded to a capture levelof 84RU. The analyte (recombinant human CD38 his tagged) was thenassociated in analysis buffer from a 2-fold dilution starting at 200 nMwith a lowest concentration of 0.78 nM for 6 minutes. Dissociation wasperformed in analysis buffer over 10 minutes. Regeneration steps betweensample concentrations were performed in 3M MgCl₂, three times for 0.5minutes. A flow rate of 25 μl/min was maintained throughout the process.Kinetics data were fit using a global fit on the analysis softwareprovided by Biacore with reference subtraction.

Avidity binding measurements of anti-CD38 Antibodies: Ni-NTA sensorswere equilibrated off-line in assay buffer for 30 minutes and thenmonitored on-line for 60 seconds for baseline establishment. They wereloaded with 4.2 nM antigen (recombinant human CD38 HIS tagged) for 50minutes, afterwards they were transferred to assay buffer for 0.5minutes for wash and again for 1 min in assay buffer for base linedetermination. Then the antibody was associated at differentconcentrations (as described in FIGS. 8) for 50 min. Afterwards theywere transferred to assay buffer for 30 minutes for off-ratemeasurement. Kinetics data were fit using a 1:1 binding model in thedata analysis software provided by ForteBio.

Epitope Binning: Epitope binning of antibodies can be performed on aForte Bio Octet Red384 system (Pall Forte Bio Corp., USA) using astandard sandwich binning assay. The anti-human CD38 antibody can beloaded onto AHQ sensors and unoccupied Fc-binding sites on the sensorand blocked with a non-relevant human IgG1 antibody. Sensors can beexposed to 100 nM target antigen followed by a second anti-CD38antibody, the reference monoclonal agonistic mouse anti-human CD38antibodies (IB4). Data can be processed using Forte Bio Data AnalysisSoftware 7.0. Additional binding by a second antibody after antigenassociation indicates an unoccupied epitope, while no binding indicatesepitope blocking.

Binding of anti-CD38 Antibodies to CD38-expressing cells: The candidatehits are evaluated by binding to lymphoblast like human cell lines,Ramos and Daudi. Binding to CD38 expressing human cell lines (Ramos andDaudi) was examined by staining test articles (anti-CD38 primaryantibodies) with 20 μg/ml antibodies followed by semi-log dilutionseries (7-point) for 30 minutes on ice. This was followed by stainingwith a secondary antibody (rabbit anti-human Fcg F(ab′)2) concentrationof 5 μg/ml for 30 minutes on ice. All samples were stained intriplicates. To minimize cross-linking induced cell death mediated bybinding of the secondary antibody, cell lines were examined in stainingcohorts of 4 test articles at a time. Live cells were gated using FSC vsSSC parameters by flow cytometry during sample acquisition. Meanfluorescence intensity (MFI) of stained cells were plotted on an XYchart, graphing MFI against the log of the concentration, and the datafit to a non-linear regression curve from which the EC50 is calculated.

Recloning, producing, and characterizing of aCD38-a-309 as human IgG1expressed in mammalian cells: Synthesis of codon optimized VH and VLcoding sequences for the antibody was performed by Genewiz. cDNAs ofvariable regions were cloned into the antibody expression vector(Icosagen, EST) containing human IgG1 heavy chain and kappa light chainconstant regions (P01857 and P01834 respectively). Full length heavy andlight chain cDNAs were verified by sequencing in final vectors and thenrecloned for expressing them using the QMCF Technology (Icosagen) astable episomal expression system that uses CHO-based cells(CHOEBNALT85) and appropriate vectors for production of recombinantproteins, antibodies, CHOEBNALT85 cells were transfected with 1 μg ofthe expression plasmids for antibody production. 48 h after thetransfection 700 μg/ml of G418 was added to select plasmid containingcell population. For the production, temperature was shifted to 30° C.and the cultures were additionally fed. At the end of the production theculture supernatants were clarified by centrifugation (1000 g, 30minutes, and 15° C.), PMSF was added and supernatants were processed orfrozen until purification. hIgG1 antibodies were purified by MabSelectSuRe affinity chromatography followed by Superdex 200 gel filtrationinto either PBS or PBS 100 mM L-Arg. Human IgG1 antibodies produced inCHOEBNALT85 cells were characterized for affinity towards recombinanthuman CD38, cross reactivity towards murine, rat, rabbit and cyno CD38using recombinant rabbit CD38 (65003-T08H-20; Sino Biological) andrecombinant rat CD38 (80229-R08H-20; Sino Biological).

Anti-human CD38 Ab Competition Assays: Antibody competitions areperformed on a Forte Bio Octet Red96 system (Pall Forte Bio Corp., USA)using a standard sequential binding assay. 0.625 ug/mL of recombinanthuman CD38his tagged is loaded onto Ni-NTA Biosensors for 300 s. Afterwash for 15 s and a base line step for 60 s on kinetic buffer sensorsare exposed to 66.6 nM of first antibody (Daratumumab) for 600 sfollowed by a second anti-CD38 antibody (Daratumumab (control) oraCD38-a-309) (also at 66.6 nM for 600 s). Data is processed using ForteBio Data Analysis Software 9.0. Additional binding by a second antibodyindicates an unoccupied epitope (no competition for the epitope), whileno binding indicates epitope blocking (competition for the epitope).

Results

Monoclonal antibodies (mAb) binding to recombinant human CD38extracellular protein sequence (rhCD38) have been isolated using ayeast-based antibody presentation library as described in the Materials& Methods. These antibodies were sequenced and unique clones wereproduced in yeast cells (Barnard GC et al., 2010). The cell culturesupernatants for each yeast clone expressing a unique antibody sequencewas screened for rhCD38 binding.

Based on the binding to rhCD38, sequence uniqueness and expressionlevels a panel of mAbs was identified. These antibodies were furthercharacterized for binding to recombinant Cynomolgus monkey and mouseCD38 extracellular domain protein sequences. The clones werecharacterized showing IgG binding values to monovalent rhCD38 and/orrecombinant cyno CD38 extracellular protein sequences that are comprisedbetween 10⁻⁸ M and 10⁻¹⁰M.

The KD values (for affinity and avidity measurements) and cross binninganalysis for selected antibodies are provided in Table 1:

TABLE 1 IgG Kd Human Cross- CD38-HIS binning (M) Monovalent AvidityAntibody group (affinity - Octet) (Kd) Isotype aCD38-a-309-IgG4 D3.29E−08 1.34E−10 IgG4 Daratumumab F 8.28E−08 1.80E−10 IgG1

Binding of anti-CD38 antibodies to recombinant monovalent human CD38measured by Octet binding and compared to Daratumumab, is shown in FIG.7 and FIGS. 8A and B. Binding affinity of anti-CD38 antibodies (IgG1) torecombinant monovalent human CD38 was also measured by Biacore (FIG.15), a Kd of 0.65 nM was determined. The antibody clones were alsoevaluated at the level of binding to human cells strongly expressingCD38, such as lymphoblast-like, Raji cells by flow cytometry, usingCHO-S cells as negative control. Further, binding to Daudi and Ramoscells (FIG. 3A) was confirmed.

Finally, in order to eliminate antibody sequences that would be prone toaggregation and non-specific interaction, the antibodies were screenedin a Poly Specific Reagent (PSR) assay and Affinity-CaptureSelf-Interaction Nanoparticle Spectroscopy (AC-SINS), an approach thatallows high-throughput screening for early-stage antibody development(Liu Y et al., 2014). None of the selected antibodies scored positive inthe latter assays and as such were not removed from the panel.

Among the selected hits that were sequenced and characterized asdescribed above, the clone aCD38-a-309 is an antibody presenting novelcomplementarity determining regions (CDRs; FIG. 2) binds human CD38extracellular protein sequences with a Kd value in the 10⁻⁸M range.

Thus, the aCD38-a-309 sequences (FIG. 2A) identify antibodies thatspecifically bind CD38, and whose agonistic activities associated to thefunctional features defining CD38 Modulating Antibody Agents, as thatterm is used herein, can be functionally evaluated by cell-based assaysor animal models. At this scope, aCD38-a-309-based antibody libraries inwhich either one or both residues are substituted and, among all suchmutants, only a limited number of them maintained the binding to humanCD38, for example as expressed on the surface of Daudi cells (seeExample 2). In this manner, the corresponding aCD38-a-309-based antibodyvariants can be tested as maintaining full properties of CD38 ModulatingAntibody Agents, or simply having CD38 binding properties. Furthervalidation of these variants may be pursued by using the assaysdisclosed in the Examples.

Example 2 Cell-Based Models for Validating CD38 Modulating AntibodyAgents Materials & Methods

In vitro T cell activation assay: Previously frozen primary human pan Tcells (Stemcell Technologies) were labelled with eFluor450 fluorescentdye (Life Technologies) and incubated for 72 hrs in 96-well platespre-coated with anti-CD3 antibody (0.1 μg/ml coating concentration,clone OKT3, eBiosciences) and anti-CD38 modulating antibodies coated atconcentrations of 10, 5 and 2.5 μg/ml in RPMI 1640 (Life Technologies)containing 10% FBS (Sigma), 2 mM L-Glutamine (Life Technologies) and10,000 U/mI Pen-Strep (Sigma). Readout of T cell proliferation was doneby acquisition on the flow cytometer, excluding dead cells labelled witha viability dye (Zombie NIR, BioLegend) and discriminating surfacemarkers by staining with fluorochrome labelled antibodies (CD8-FITCclone HIT8a eBiosciences, CD25-PE clone M-A251 Biolegend, CD4-BV510clone RPA-T4 BioLegend, CD38-PE-Cy7 clone HB_7, eBiosciences, CD137-APCclone 4B4-1 BioLegend). Cytokine analysis in supernatants was conductedusing the Meso Scale Discovery MSD platform, determining the expressionof IFNg, IL2, IL10, TNFa, and GM-CSF according to the manufacturer'sinstructions (Multiplex assay kits, Meso Scale Discovery; asterisk infigure indicates values above fit curve range).

In vitro NK cell activation assay. Anti-CD38 antibodies as describedherein can be characterized using an in vitro NK cell activation assayin which, for example, human PBMC are labelled with Cell Trace violetproliferation dye (Life Technologies) and cultured in the presence ofMDA-MB-231 cells in a ratio of 100:1 (culture medium IMDM, LifeTechnologies, 10% human serum heat-inactivated, Sigma, 10,000 U/mlPen-Strep, Sigma) for 5 days. Anti-CD38 antibody is added or controlcells are left untreated. Readout for proliferation quantified bydilution of fluorescent dye is done by FACS analysis. Cells are labelledwith fluorochrome conjugated antibodies and NK cells are gated byexcluding dead cells (Zombie NIR dye, Biolegend), gating on CD45+hematopoietic cells (CD45-PE-Cy7, Biolegend), further gating on CD3negative CD56 positive cells (CD56-BV711 clone H130 Biolegend; CD3-BV510clone OKT3 Biolegend).

In vitro ADCP assay: Anti-CD38 antibodies as described herein can becharacterized using an in vitro ADCP assay in which, for example,antibody-dependent cell-mediated phagocytosis (ADCP) can be performedusing in-vitro differentiated Tregs as target cells and monocyte-derivedmacrophages as the effector cells. Different effector to target ratiosare evaluated. Target cells are added at 1×10⁴ cells/well while theeffector cells are added at (1×10⁴, 2.5×10⁴, 5×10⁴ or 1×10⁵ cells/well).Anti-human CD38 antibodies are evaluated at 3 concentrations (1 μg/ml;10 μg/ml and 50 μg/ml). The assay is performed using the followingprotocol: PBMC are isolated from leucocyte cones by Ficoll gradientcentrifugation. CD14+ cells are isolated using CD14 Microbeads (CDK006,Miltenyi Biotec). Monocytes are cultured for 7 days in the presence of50 ng/ml M-CSF in RPMI 1640 (Life Technologies) containing 10% FBS(Sigma), 2mM L-Glutamine (Life Technologies) and 10,000 U/mI Pen-Strep(Sigma), fresh media containing M-CSF is added after 4 days. RegulatoryT cells (Treg) are isolated using the Human Treg Cell DifferentiationKit (130-050-201, R&D Systems). These cells are incubated in a 37° C.,5% CO₂ humidified incubator for 5 days. At day 7 macrophages andeFluor450 labelled (eBiosciences) Tregs are cocultured overnight inratios described above in the presence of CD38 or control antibodies.Phagocytosis of Tregs is determined by flow cytometry gating on CD14+cells (stained with CD14-PE-Cy7 clone MfP9 BD Biosciences) positive forthe Treg label (eFluor450 dye).

In vitro ADCC assay: Antibody-dependent cell-mediated cytotoxicityassays (ADCC assays) were performed for the characterization ofanti-human CD38 antibodies using Daudi (CD38 positive) human cell lineas a target cell with human PBMC as the source of effector cells.Effector to Target ratios would be evaluated at 50 to 1 or 25 to 1 withtest articles (anti-CD38 primary antibodies) to be evaluated with topconcentration of 10 μg/ml followed by a log series (7 points) intriplicate for 4 hours at 37° C. 5% CO2. PBMCs were primed with IL-2 andIL-2 was present during co-culture assays. Prior to in-vitro culture,target cell lines were labelled with 1 μM Calcein AM and incubated with2.5 mM probenecid. Lysed cells release the loaded Calcein into thesupernatant, which allows for fluorescent measurement. Calcein AMrelease was analysed by excel and GraphPad software analysis to generatedose response curves by normalization where 1% saponin treatment valueswill be used to determine maximal lysis. Percentage target cell lysiswas plotted on an XY chart, graphing normalized Calcein AM percentagerelease against the log of the concentration, and the data fit to ano-linear regression curve from which the EC50 was calculated.

In vitro CDC assay: CDC activity to CD38 expressing human cell lines(Daudi) was examined by treating cells with test articles (anti-CD38primary antibodies) at a top concentration of 10μg/ml followed by a logdilution series (7 points) in triplicate with a final concentration of10% normal human serum complement. Samples were cultured for 3 hours at37° C. 5% CO2. Following culture conditions, cells washed andre-suspended in 1× PBS with propidium iodide (PI) at a finalconcentration of 5 μg/ml prior to flow cytometry analysis. Total cellswere examined by flow cytometry during sample acquisition. Percentage ofPI positive cells were plotted on an XY chart, graphing percentage PIagainst the log of the concentration, and the data fit to a non-linearregression curve from which the EC50 is calculated.

In vitro ADCP reporter assay: ADCP activity to CD38 expressing humancell lines was examined by using the Promega Bioassay core kit G9901.5000 Raji cells/well of target wells were plated in 25 ul medium perwell using a 96 well white polystyrene plate (Costar Cat#3917). Testantibodies were serial diluted 1:3 in a separate plate. 25 ul serialdiluted antibody was added to the cells. 50000 cells/well of theeffector cells were added to the plate (25 ul/well). Plates wereincubated over night for 20hours at 37 C. The next day the plate wasremoved from the incubator and kept at room temperature for 20 minutes.60 ul Bio-Glo Luciferase assay substrate were added to each well,incubated for 30 min. Luminsence was read using the GloMax MultiDetection System. Cell culture medium: RPMI+4% Low IgG Serum.

Direct cell death assay: Direct proapoptotic activity to CD38 expressinghuman cell lines (Daudi) was examined by treating cells with testarticles (anti-CD38 primary antibodies) at a top concentration of 10μg/ml followed by a log dilution series (7 points) in triplicate. Celldeath by Fcγ receptor-mediated cross-linking activity was examined bytreating cells with test articles (anti-CD38 primary antibodies(at a topconcentration of 10 μg/ml followed by a log serial dilution (7 points)in triplicate followed by 5 μg/ml rabbit anti-human Fcγ F(ab′)2(secondary antibody). Samples were cultured for 24 hours at 37° C. 5%CO₂. Following culture conditions, cells were washed and resuspended inAnnexin V binding buffer and 7-AAD to examine cell death by flowcytometry analysis. Total cells were examined by flow cytometry duringsample acquisition. Percentage of late apoptotic cells were plotted onan XY chart, graphing percentage Annexin V-positive and 7-AAD-positivecells against the log of the concentration and the data fit to anon-linear regression from which the EC50 is calculated.

Enzymatic activities of CD38 on the cell surface (cyclase andNADase/hydrolase activities): both cyclase and NADase activity of CD38were measured on the cell surface of Daudi cells and in Jurkat cells bymonitoring the CD38-dependent conversion of NGD+ (Sigma Aldrich) andE-NAD+ (Sigma Aldrich) into their respective fluorescent products: cGDPR(cyclic product from NDG+) and 5′-eAMP (hydrolysis product of E-NAD+).150 thousand Daudi or Jurkat cells were incubated for 20 minutes on icewith 10 μg/ml antibodies in 75 μl of PBS (Thermo Fisher); after 20minutes, 75 μl of enzymatic reaction buffer (or control buffers) wereadded and the cells were incubated at 37° C. for 45 for Daudi cells and60 minutes for Jurkat cells. The enzymatic reaction buffer included 20mM UltraPure Tris-HCl Buffer (Thermo Fisher), pH 7.5 in PBS, (ThermoFisher), 200 μM of either NGD+ or E-NAD+, 20 μM dipyridamole (nucleosidetransporter inhibitor) and 20 μM b-g-meATP (CD203a/PC-1 inhibitor).After the incubation at 37° C., the cells were pelleted by mean ofcentrifugation at 550×g and 100 μl of supernatant were utilised forfluorescence measurements in a Molecular Device SpectraMax MiniMax 300plate reader (excitation wavelength 300 and emission wavelength 410).

Statistics. Prism software (GraphPad) was used to perform curve fittingand to determine EC50 values and maximal activity.

Results

The EC50 values and percentage lysis results from the ADCC and CDCassays are shown in Tables 2 and 3:

TABLE 2 ADCC EC50 Max. lysis Antibody ug/ml % aCD38-a-309 0.0072-0.00936-68 Daratumumab  0.004-0.0139 36-68 Isotype hIgG1 n/a —

TABLE 3 CDC: EC50 Max. lysis Antibody ug/ml % aCD38-a-309 0.04-0.1997-97.2 Daratumumab 0.07-0.11 91.3-92    Isotype hIgG1 n/a —

The aCD38-a-309 candidate antibody, as other antibodies that have beencharacterized in Example 1, has been further evaluated with respect toimmune cells. In a first series of experiments, aCD38-a-309 shows dosedependent binding to human lymphoblast like cells (Daudi and Ramos celllines) (FIG. 3). When tested using T cells, for instance whenaCD38-a-309 is used for coating a plate for culturing such cells,aCD38-a-309 strongly increases human T cell activation while thereference anti-CD38 antibody (DARA) is displaying much weaker agonistactivity (FIG. 4A). The agonist activity of aCD38-a-309 is furtheremphasized by stronger proinflammatory cytokine secretion by human Tcells triggered by aCD38-a-309 when compared to DARA (FIG. 4B).aCD38-a-309 and DARA show comparable activity in ADCC, CDC, and ADCPassays (FIG. 5A and 5B and FIG. 9). DARA and aCD38-a-309 show alsocomparable activity in direct killing of CD38 expressing tumour cells(Daudi)(FIG. 5C). In addition to the killing activities in vitro,aCD38-a-309 and DARA both inhibit cyclase activity of CD38 (FIG. 6A).aCD38-a-309 and DARA do not affect the NADase (NAD+ hydrolase) activityof CD38 (FIG. 6B) in Daudi cell assay. In Jurkat cell assay bothaCD38-a-309 and DARA inhibited cyclase activity of CD38 (FIG. 10A).Whilst aCD38-a-309 inhibited NADase (NAD+ hydrolase) activity of CD38,and DARA stimulated NADase (NAD+ hydrolase) activity of CD38 in theJurkat cell assay (FIG. 10B). In conclusion, aCD38-a-309 has beencharacterized as an exemplary anti-CD38 antibody that presents theactivities of a CD38 Modulating Antibody Agent with respect to immunecells in different experimental set ups.

Example 3 Validation of CD38 Modulating Antibody Agent in Animal Models

Materials & Methods

Lymphoma cells-based models: Anti-CD38 antibodies as described hereincan be characterized using animal models in which, for example, Raji andRamos tumor cells are cultured in RPMI 1640 containing 2 mM L-glutaminesupplemented with 10% fetal bovine serum+1 mM Na Pyruvate+4.5 g/LGlucose+10 mM Hepes. Healthy female cb17 SCID mice are obtained fromCharles River. Tumors are induced by intravenous injection of 5×10⁶ Rajicells or 1×10⁶ Ramos cells in 200 μL of RPMI 1640 into the caudal veinof the animals. Cell injection is performed 24 to 72 hours after a wholebody irradiation with a γ-source (1.44 Gy/mouse, 60Co, BioMep,Bretenières, France). Mice are randomized into treatment groups bybodyweight, 8 mice per group. In all models animals from group 1 receiveintravenous injections of vehicle at 5 ml/kg twice a week for threeconsecutive weeks (TWx3). Animals from group 2 receive intravenousinjections of DARA at 10 mg/kg/inj. twice a week for three consecutiveweeks (TWx3). Animals from group 3 receive intravenous injections ofaCD38-a-309 at 10 mg/kg/inj. twice a week for three consecutive weeks(TWx3). Mice are sacrificed after a maximum of 8 weeks.

Solid Tumor Model:

Female CB17SCID mice were injected with 1×10⁷ Ramos tumour cells in 0%Matrigel subcutaneously in the flank, n=10 per group. Treatment startedwhen tumours reached 100-130 mm³ size for twice a week for three weeks.Mice were treated with 10 mg/kg intra venously with the antibodyaCD38-a-309 compared to daratumumab and a vehicle control. Mice weresacrificed when the tumour volume reached 2000 mm³ or 60 days, whichever was later.

Results

The therapeutic properties of aCD38-a-309 can be tested in animal modelfor human cancer, in particular using immunocompromised mice where theproperties of a CD38 Modulating Antibody Agent with respect to thekilling of human tumor cells can be more appropriately evaluated. Theseproperties, not only in terms of animal survival but also withconcurrent immunological effects can be further investigated in other invivo models for human tumors (in particular solid cancers) that arebased on the injection with either human cancer lines or human primarycancer cells, in which solid tumors grow subcutaneously, as described inthe literature (Morton J J et al. 2016; Holzapfel B M et al., 2015).

aCD38-a-309 showed enhanced anti-tumour activity against subcutaneouslyinjected Ramos cells when compared to daratumumab (FIG. 11). aCD38-a-309also showed equivalent anti-tumor activity against intravenouslyinjected Ramos and Raji cells when compared to daratumumab (FIG. 12 andFIG. 13).

Properties can be further investigated in ex vivo models based on theuse of tumour samples directly isolated from patients from which tumourcells and immune cells are isolated and tested in vitro for theirresponse to the anti-CD38 antibodies, as measured by cell activation,proliferation, cytokine production and/or cell death. Additionalfeatures such changes in gene expression in selected tissues orbiological materials can be evaluated, possibly by administeringaCD38-a-309 in different doses and/or in combination with otheranti-cancer agents (such as inhibitors of kinases or of other enzymes,antibodies, radio/chemo-therapy, adjuvants, or vaccines).

Example 4 Antibody Binding to Mutant CD38

Materials and Methods: Two mutant version of human CD38 wereconstructed. In one version D was mutated to G at position 202 (D202G)and in the second version S was mutated to F at position 274 (S274F).

The binding of aCD38-a-309 to each of the mutated CD38 proteins wasassessed, and compared to daratumumab.

Results

TABLE 4 Binding Reactivity (% WT) Antibody Mutation - D202G Mutation -S274F aCD38-a-309 Fab 10.9 (10) 119.2 (4) Daratumumab 39.9 (8)   6.8 (6)The results showed that binding of aCD38-a-309 was not affected by theintroduction of mutation S274F into human CD38 but was affected by theintroduction of mutant D202G. This compares to Daratumumab whereantibody binding was affected by the introduction of mutation S274F.These results support aCD38-a-309 binding to a different epitope thandaratumumab.

A summary of the sequences included in the application is providedbelow:

Description of SEQ ID NO Antibody Sequences Also referred to as: 1aCD38-a-309 variable aCD38-a-309-HCDR1 heavy chain CDR1 2 aCD38-a-309variable aCD38-a-309-HCDR2 heavy chain CDR2 3 aCD38-a-309 variableaCD38-a-309-HCDR3 heavy chain CDR3 4 aCD38-a-309 variableaCD38-a-309-HCDR123 heavy chain CDR 1, 2, 3 and FR 2, 3, 4 5 aCD38-a-309variable aCD38-a-309-LCDR1 light chain CDR1 6 aCD38-a-309 variableaCD38-a-309-LCDR2 light chain CDR2 7 aCD38-a-309 variableaCD38-a-309-LCDR3 light chain CDR3 8 aCD38-a-309 variableaCD38-a-309-LCDR123 light chain CDR 1, 2, 3 and FR 2, 3, 4 9 Human CD38Uniprot sequence P28907 10 aCD38-a-309 variable aCD38-a-309-VH heavychain CDR 1, 2, 3 and FR 1, 2, 3, 4 11 aCD38-a-309 variableaCD38-a-309-VL light chain CDR 1, 2, 3 and FR 1, 2, 3, 4 12 aCD38-a-309variable heavy chain CDR 1, 2, 3 and FR 2, 3 13 aCD38-a-309 variablelight chain CDR 1, 2, 3 and FR 2, 3 14 Daratumumab variable heavy chain15 Daratumumab variable light chain

Equivalents and Scope

Those skilled in the art will appreciate that the present invention isdefined by the appended claims and not by the Examples or otherdescription of certain embodiments included herein.

Similarly, the singular forms “a”, “an”, and “the” include pluralreferents unless the context clearly dictates otherwise.

Unless defined otherwise above, all technical and scientific terms usedherein have the same meaning as commonly understood by one of ordinaryskill in the art to which this invention belongs. Any methods andmaterials similar or equivalent to those described herein can also beused in the practice or testing of the present invention. Generally,nomenclatures used in connection with, and techniques of, cell andtissue culture, molecular biology, immunology, genetics and protein andnucleic acid chemistry described herein are those well known andcommonly used in the art, or according to manufacturer's specifications.

All publications mentioned herein are incorporated herein by referenceto disclose and describe the methods and/or materials in connection withwhich the publications are cited.

REFERENCES

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The invention claimed is:
 1. An antibody or antigen-binding fragmentthereof that binds specifically to CD38, comprising a) aCD38-a-309-HCDR3amino acid sequence (SEQ ID NO: 3) as a variable heavy chaincomplementarity determining region 3; b) aCD38-a-309-HCDR1 amino acidsequence (SEQ ID NO: 1) as a variable heavy chain complementaritydetermining region 1; c) aCD38-a-309-HCDR2 amino acid sequence (SEQ IDNO: 2) as a variable heavy chain complementarity determining region 2;d) aCD38-309-LCDR1 amino acid sequence (SEQ ID NO: 5) as a variablelight chain complementarity determining region 1; e) aCD38-a-309-LCDR2amino acid sequence (SEQ ID NO: 6) as a variable light chaincomplementarity determining region 2; and f) aCD38-a-309-LCDR3 aminoacid sequence (SEQ ID NO: 7) as a variable light chain complementaritydetermining region
 3. 2. The antibody or antigen-binding fragmentthereof according to claim 1, wherein the antibody or antigen-bindingfragment thereof comprises a variable heavy chain sequence having atleast 95% sequence identity to aCD38-a-309-HCDR123 amino acid sequenceas set forth in SEQ ID NO:
 4. 3. The antibody or antigen-bindingfragment thereof according to claim 1, wherein the antibody orantigen-binding fragment thereof comprises a variable light chainsequence having at least 95% sequence identity to aCD38-a-309-LCDR123amino acid sequence as set forth in SEQ ID NO:
 8. 4. The antibody orantigen-binding fragment thereof according to claim 1 wherein theantibody or an antigen-binding fragment thereof is selected from thegroup consisting of: a) an antibody or antigen binding fragment thereofcomprising a heavy chain variable region comprising the sequence of SEQID NO: 4 and a light chain variable region comprising the sequence ofSEQ ID NO: 8; b) an antibody or antigen binding fragment thereofcomprising a heavy chain variable region comprising the sequence of SEQID NO: 10 and a light chain variable region comprising the sequence ofSEQ ID NO: 11; and c) an antibody or antigen binding fragment thereofcomprising a heavy chain variable region comprising the sequence of SEQID NO: 12 and a light chain variable region comprising the sequence ofSEQ ID NO:
 13. 5. The antibody or an antigen-binding fragment thereofaccording to claim 1 wherein the antibody or antigen-binding fragmentthereof is a-fucosylated.
 6. The antibody or antigen-binding fragmentthereof according to claim 1, wherein the antibody or antigen-bindingfragment thereof is a monoclonal antibody, a domain antibody, a singlechain antibody, a Fab fragment, a F(ab')2 fragment, a single chainvariable fragment (scFv), a scFv-Fc fragment, a single chain antibody(scAb), or a single domain antibody.
 7. The antibody or antigen-bindingfragment thereof of according to claim 1, wherein the antibody orantigen-binding fragment thereof is a rabbit, mouse, chimeric, humanizedor fully human antigen-binding antibody.
 8. The antibody orantigen-binding fragment thereof according to claim 1, wherein theantibody or antigen-binding fragment thereof is selected from the groupconsisting of IgG1, IgG2, IgG3, and IgG4 isotype antibodies.
 9. Theantibody or antigen-binding fragment thereof according to claim 1,wherein the antibody or antigen-binding fragment thereof is comprised ina bispecific antibody, a multispecific antibody, or an immunoconjugatefurther comprising a therapeutic or diagnostic agent.
 10. The antibodyor antigen-binding fragment thereof according to claim 1, wherein theantibody or antigen-binding fragment thereof binds the extracellulardomain of human CD38.
 11. The antibody or antigen-binding fragmentthereof according to claim 1, wherein the antibody or antigen-bindingfragment thereof binds cells expressing human CD38 on their surface andis a CD38 Modulating Antibody Agent.
 12. A nucleic acid moleculeencoding the antibody or antigen-binding fragment thereof according toclaim
 1. 13. A nucleic acid vector comprising the nucleic acid moleculeof claim cm
 12. 14. A host cell comprising the nucleic acid vector ofclaim
 13. 15. A method for producing an antibody or antigen-bindingfragment thereof comprising a) aCD38-a-309-HCDR3 amino acid sequence(SEQ ID NO: 3) as a variable heavy chain complementarity determiningregion 3; b) aCD38-a-309-HCDR1 amino acid sequence (SEQ ID NO: 1) as avariable heavy chain complementarity determining region 1; c)aCD38-a-309-HCDR2 amino acid sequence (SEQ ID NO: 2) as a variable heavychain complementarity determining region 2; d) aCD38-a-309-LCDR1 aminoacid sequence (SEQ ID NO: 5) as a variable light chain complementaritydetermining region 1; e) aCD38-a-309-LCDR2 amino acid sequence (SEQ IDNO: 6) as a variable light chain complementarity determining region 2;and f) aCD38-a-309-LCDR3 amino acid sequence (SEQ ID NO: 7) as avariable light chain complementarity determining region 3, the methodcomprising culturing a host cell of claim
 14. 16. A compositioncomprising an antibody or antigen-binding fragment thereof according toclaim 1 and a pharmaceutically acceptable carrier or excipient.
 17. Akit comprising the composition of claim 16 in a container.